Interferon-alpha polypeptides and conjugates

ABSTRACT

The present invention provides interferon-alpha polypeptides and conjugates, and nucleic acids encoding the polypeptides. The invention also includes compositions comprising these polypeptides, conjugates, and nucleic acids; cells containing or expressing the polypeptides, conjugates, and nucleic acids; methods of making the polypeptides, conjugates, and nucleic acids; and methods of using the polypeptides, conjugates, and nucleic acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119(e), this application claims the benefit ofU.S. Provisional Application Ser. No. 60/572,504 filed on May 19, 2004,the disclosure of which is incorporated by reference herein in itsentirety for all purposes.

FIELD OF THE INVENTION

The present invention relates generally to polynucleotides andpolypeptides encoded therefrom, conjugates of the polypeptides, as wellas vectors, cells, antibodies, and methods for using and producing thepolynucleotides, polypeptides, and conjugates.

BACKGROUND OF THE INVENTION

Interferon-alphas are members of the diverse helical-bundle superfamilyof cytokine genes (Sprang, S. R. et al. (1993) Curr. Opin. Struct. Biol.3:815-827). The human interferon-alphas are encoded by a family of over20 tandemly duplicated nonallelic genes and psuedogenes that share85-98% sequence identity at the amino acid level (Henco, K. et al.(1985) J. Mol. Biol. 185:227-260). Genes which express activeinterferon-alpha proteins have been grouped into 13 families accordingto genetic loci. Known expressed human interferon-alpha proteins andtheir allelic variations are tabulated in Allen G. and Diaz M. O. (1996)J. Interferon and Cytokine Res. 16:181-184.

Interferon-alphas have been shown to inhibit various types of cellularproliferation, and are especially useful for the treatment of a varietyof cellular proliferation disorders frequently associated with cancer,particularly hematologic malignancies such as leukemias. These proteinshave shown antiproliferative activity against multiple myeloma, chroniclymphocytic leukemia, low-grade lymphoma, Kaposi's sarcoma, chronicmyelogenous leukemia, renal-cell carcinoma, urinary bladder tumors andovarian cancers (Bonnem, E. M. et al. (1984) J. Biol. Response Modifiers3:580; Oldham, R. K. (1985) Hospital Practice 20:71).

Interferon-alphas are also useful against various types of viralinfections (Finter, N. B. et al. (1991) Drugs 42(5):749).Interferon-alphas have activity against human papillomavirus infection,Hepatitis B, and Hepatitis C infections (Finter, N. B. et al., 1991,supra; Kashima, H. et al. (1988) Laryngoscope 98:334; Dusheiko, G. M. etal. (1986) J. Hematology 3 (Supple. 2):S199; Davis, G L et al. (1989) N.England J. Med. 321:1501). The role of interferons and interferonreceptors in the pathogenesis of certain autoimmune and inflammatorydiseases has also been investigated (Benoit, P. et al. (1993) J.Immunol. 150(3):707).

Although these proteins possess therapeutic value in the treatment of anumber of diseases, they have not been optimized for use aspharmaceuticals. For example, dose-limiting toxicity, receptorcross-reactivity, and short serum half-lives significantly reduce theclinical utility of many of these cytokines (Dusheiko, G. (1997)Hepatology 26:112S-121S; Vial, T. and Descotes, J. (1994) DrugExperience 10:115-150; Funke, I. et al. (1994) Ann. Hematol. 68:49-52;Schomburg, A. et al. (1993) J. Cancer Res. Clin. Oncol. 119:745-755).Diverse and severe side effect profiles which accompany interferonadministration include flu-like symptoms, fatigue, hallucination, fever,hepatic enzyme elevation, and leukopenia (Pontzer, C. H. et al. (1991)Cancer Res. 51:5304; Oldham, 1985, supra).

Hepatitis-C virus (HCV) is a nonhost integrated RNA virus with a veryhigh rate of replication and is therefore associated with a large degreeof genetic diversity. At least six genotypes and more than thirtysubtypes of HCV RNA have been identified. HCV genotype has been shown tobe a predictor of response to IFN-alpha therapy. Patients infected withHCV genotypes 2 and 3 have been found to generally respond well tointerferon therapy. Patients infected with genotypes 4, 5 and 6 tend torespond less well. Patients infected with HCV genotype 1 tend to respondvery poorly to interferon therapy, with about 50% of Genotype 1 patientsclassified as “nonresponders” towards IFN-alpha therapy. Genotype 1 iscurrently the most prevalent form of Hepatitis C, infectingapproximately 70% of patients in the US and 50% of patients in Europe.Clearly, there is a pressing need for more effective therapies for HCVinfection, particularly of the Genotype 1 variety.

There is genetic and biochemical evidence that Genotype 1 HCV (and othersubtypes) actively attenuate the IFN-alpha signaling pathway byinhibiting key IFN responsive proteins such as the dsRNA-activatedserine/threonine protein kinase PKR (Katze M. G., et al. (2002) Nat.Rev. Immunol. 2(9):675-687). As a likely consequence of this geneticdiversity and active inhibition of the antiviral response, HCV(particularly Genotype 1) has the ability to escape the host's immunesurveillance, leading to a high rate of chronic infection. The extensivegenetic heterogeneity of HCV has important diagnostic and clinicalimplications, potentially accounting for variations in clinical course,difficulties in vaccine development, and lack of response to therapy.

The present invention addresses the need for interferon-alpha moleculeswhich exhibit enhanced antiviral and/or immunomodulatory efficacy. Theinvention provides novel interferon-alpha polypeptides and polypeptideconjugates, nucleic acids encoding the polypeptides, and methods ofusing such molecules. Such molecules would be of beneficial use in avariety of applications, including, e.g., therapeutic and prophylactictreatments, particularly for viral infections and diseases andconditions associated with viral infections. The present inventionfulfills these and other needs.

SUMMARY OF THE INVENTION

The present invention provides novel polypeptides, including variantsthereof and fusion proteins comprising such polypeptides. The inventionalso provides conjugates comprising a polypeptide of the inventioncovalently linked to one or more non-polypeptide moieties. The inventionalso provides nucleic acids encoding any of the polypeptides of theinvention, and vectors and host cells comprising such nucleic acids. Inaddition, the invention provides methods of making and using suchpolypeptides, conjugates, and nucleic acids, and other features apparentupon further review.

In one aspect, the invention provides an isolated or recombinantpolypeptide, the polypeptide comprising a sequence identified as one ofSEQ ID NOs:2-35 and SEQ ID NOs: 37-44 (such as one of SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO: 10, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, and SEQID NO:44). The invention also provides fusion proteins and conjugatescomprising any of these polypeptides, nucleic acids encoding thesepolypeptides, and methods of making and using these polypeptides.

The invention also provides isolated or recombinant polypeptides whicheach comprise a sequence which (a) differs in 1 to 16 amino acidpositions from SEQ ID NO:1 and (b) comprises one or more substitutionsrelative to SEQ ID NO:1 selected from the group consisting of F48A/L;V51P; F55A; F65A; F68P; L111A; and V114P. Some such polypeptidescomprise a sequence which differs from SEQ ID NO:1 in 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid positions, e.g., in1-15 amino acid positions, in 1-14 amino acid positions, in 1-13 aminoacid positions, in 1-12 amino acid positions, 1-11 amino acid positions,in 1-10 amino acid positions, in 1-9 amino acid positions, in 1-8 aminoacid positions, in 1-7 amino acid positions, in 1-6 amino acidpositions, in 1-5 amino acid positions, in 1-4 amino acid positions, in1-3 amino acid positions, or in 1-2 amino acid positions. In someinstances, the polypeptide further comprises one or more substitutionsrelative to SEQ ID NO:1 selected from the group consisting of H47Q;V51A; Q52P/E; A53T; F55S; L56V; F57L; Y58H; M61I; N113K; V114E; andE160D. In some instances, the polypeptide exhibits an interferon-alphaactivity (such as, e.g., antiviral activity, T_(H)1 differentiationactivity, and/or antiproliferative activity). The invention alsoprovides fusion proteins and conjugates comprising any of thesepolypeptides, nucleic acids encoding such polypeptides, and methods ofmaking and using such polypeptides.

The invention also provides isolated or recombinant polypeptides whicheach comprise a sequence which (a) differs in 0 to 16 amino acidpositions from SEQ ID NO:13 and (b) comprises one or more of: Ala or Leuat position 48; Pro at position 51; Ala at position 55; Ala at position65; Pro at position 68; Ala at position 111; and Pro at position 114(position numbering relative to SEQ ID NO: 13). Some such polypeptidescomprise a sequence which differs from SEQ ID NO:13 in 0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid positions, e.g., in0-15 amino acid positions, in 0-14 amino acid positions, in 0-13 aminoacid positions, in 0-12 amino acid positions, in 0-11 amino acidpositions, in 0-10 amino acid positions, in 0-9 amino acid positions, in0-8 amino acid positions, in 0-7 amino acid positions, in 0-6 amino acidpositions, in 0-5 amino acid positions, in 0-4 amino acid positions, in0-3 amino acid positions, in 0-2 amino acid positions, or in 0-1 aminoacid positions. Some such polypeptides comprise an Ala or a Leu atposition 48. Some such polypeptides comprise an Ala at position 48. Insome instances, the polypeptide further comprises one or moresubstitution(s) relative to SEQ ID NO:13 selected from the groupconsisting of H47Q; V51A; Q52P/E; A53T; F55S; L56V; F57L; Y58H; M61I;N113K; V114E; and E160D. In some instances, the polypeptide exhibits aninterferon-alpha activity (e.g., antiviral activity, T_(H)1differentiation activity, and/or antiproliferative activity). Some suchpolypeptides further comprise one or more additional amino acid(s), suchas a methionine added to the N-terminus. The invention also providesfusion proteins and conjugates comprising these polypeptides, andisolated or recombinant nucleic acids encoding these polypeptides.

The invention also provides isolated or recombinant polypeptides whicheach comprise a sequence which (a) differs in 1 to 16 amino acidpositions from SEQ ID NO:36 and (b) comprises one or more substitutionsrelative to SEQ ID NO:36 selected from the group consisting of M21A;I24P; F48A/L; T51P; S55A; F65A; F68P; F90A; M93P; L111A; V114P; F124A;I127P and E160D. Some such polypeptides comprise a sequence whichdiffers from SEQ ID NO:36 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, or 16 amino acid positions, e.g., in 1-15 amino acid positions,in 1-14 amino acid positions, in 1-13 amino acid positions, in 1-12amino acid positions, 1-11 amino acid positions, in 1-10 amino acidpositions, in 1-9 amino acid positions, in 1-8 amino acid positions, in1-7 amino acid positions, in 1-6 amino acid positions, in 1-5 amino acidpositions, in 1-4 amino acid positions, in 1-3 amino acid positions, orin 1-2 amino acid positions. In some instances, the polypeptide furthercomprises one or more substitutions relative to SEQ ID NO:36 selectedfrom the group consisting of P26L; H47Q; T51V; S55P/F; V56L; H58Y; L60M;F90Y; M93L; N95D; N113K; V114E; R125Q; T132K; and L154F. In someinstances, the polypeptide exhibits an interferon-alpha activity (suchas, e.g., antiviral activity, T_(H)1 differentiation activity, and/orantiproliferative activity). The invention also provides fusion proteinsand conjugates comprising any of these polypeptides, nucleic acidsencoding such polypeptides, and methods of making and using suchpolypeptides.

The invention also provides isolated or recombinant polypeptides whicheach comprise a sequence which (a) differs in 0 to 16 amino acidpositions from SEQ ID NO:38 and (b) comprises one or more of: Ala atposition 21; Pro at position 24; Ala or Leu at position 48; Pro atposition 51; Ala at position 55; Ala at position 65; Pro at position 68;Ala at position 90; Pro at position 93; Ala at position 111; Pro atposition 114; Ala at position 124; Pro at position 127; and Glu atposition 160 (position numbering relative to SEQ ID NO:38). Some suchpolypeptides comprise a sequence which differs from SEQ ID NO:38 in 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acidpositions, e.g., in 0-15 amino acid positions, in 0-14 amino acidpositions, in 0-13 amino acid positions, in 0-12 amino acid positions,in 0-11 amino acid positions, in 0-10 amino acid positions, in 0-9 aminoacid positions, in 0-8 amino acid positions, in 0-7 amino acidpositions, in 0-6 amino acid positions, in 0-5 amino acid positions, in0-4 amino acid positions, in 0-3 amino acid positions, in 0-2 amino acidpositions, or in 0-1 amino acid positions. Some such polypeptidescomprise an Ala or a Leu at position 48. Some such polypeptides comprisean Ala at position 48. In some instances, the polypeptide furthercomprises one or more substitution(s) relative to SEQ ID NO:38 selectedfrom the group consisting of P26L; H47Q; V51T; F55P/S; L56V; Y58H; L60M;F90Y; M93L; N95D; N113K; V114E; R125Q; T132K; and F154L. In someinstances, the polypeptide exhibits an interferon-alpha activity (e.g.,antiviral activity, T_(H)1 differentiation activity, and/orantiproliferative activity). Some such polypeptides further comprise oneor more additional amino acid(s), such as a methionine added to theN-terminus. The invention also provides fusion proteins and conjugatescomprising these polypeptides, nucleic acids encoding such polypeptides,and methods of making and using such polypeptides.

The invention also provides conjugates comprising a polypeptide of theinvention, such as any of the polypeptides of the invention describedabove, and at least one non-polypeptide moiety attached to an attachmentgroup of the polypeptide, wherein the conjugate exhibits aninterferon-alpha activity. In some instances, the non-polypeptide moietyis a polymer (for example, a polyalkylene oxide molecule, such as apolyethylene glycol (PEG), such as a monomethoxypolyethylene glycol(mPEG)), or a sugar moiety. The at least one non-polypeptide moiety may,for example, be attached to a cysteine, to a lysine, to the N-terminalamino group of the polypeptide, or to an in vivo glycosylation site ofthe polypeptide. The invention also provides methods of making and usingsuch conjugates.

The invention also provides isolated or recombinant nucleic acidsencoding any of the polypeptides of the invention. The invention alsoprovides vectors and host cells comprising such nucleic acids, andmethods of making polypeptides of the invention comprising culturinghost cells comprising such nucleic acids.

In another aspect, the invention provides a method of inhibiting viralreplication in virus-infected cells, the method comprising contactingthe virus-infected cells with a polypeptide or a conjugate of theinvention. The invention also provides a method of reducing the numberof copies of a virus in virus-infected cells, comprising contacting thevirus-infected cells with a polypeptide or a conjugate of the invention.

In another aspect, the invention provides a method of treating acondition which is responsive to interferon-alpha, comprisingadministering to a subject afflicted with the condition a compositioncomprising a polypeptide of the invention or a conjugate of theinvention in an amount effective to ameliorate a symptom associated withthe condition. Such conditions include Chronic Hepatitis C infection,Chronic Hepatitis B infection, Hairy Cell Leukemia, Malignant Melanoma,Follicular Lymphoma, Condylomata Acuminata, AIDS-related Kaposi'sSarcoma, Non-Hodgkin's Lymphoma, Chronic Melogenous Leukemia, Basal CellCarcinoma, Multiple Myeloma, carcinoid tumors, bladder cancer, Crohn'sDisease, Cutaneous T Cell Lymphoma, Renal Cell Carcinoma, MultipleSclerosis, and AIDS. The invention also includes the use of acomposition comprising a polypeptide of the invention or a conjugate ofthe invention to treat a condition which is responsive tointerferon-alpha, such as for example a condition described above.

These and other objects and features of the invention will become morefully apparent when the following detailed description is read inconjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show biphasic timecourses for viral clearance fromHCV-infected cells following IFN-alpha treatment (A. Nonresponderkinetics; B. Responder kinetics).

FIG. 2 shows an alignment of the sequence of a polypeptide of theinvention (SEQ ID NO:13) with the following human interferon-alpha(huIFN-alpha) polypeptide sequences: huIFN-alpha 1a (SEQ ID NO:45),huIFN-alpha 2b (SEQ ID NO:46), huIFN-alpha 4b (SEQ ID NO:48),huIFN-alpha 5 (SEQ ID NO:49), huIFN-alpha 6 (SEQ ID NO:50), huIFN-alpha7 (SEQ ID NO:51), huIFN-alpha 8b (SEQ ID NO:52), huIFN-alpha 10a (SEQ IDNO:53), huIFN-alpha 14a (SEQ ID NO:54), huIFN-alpha 16 (SEQ ID NO:55),huIFN-alpha 17b (SEQ ID NO:56) and huIFN-alpha 21b (SEQ ID NO:57). Thenaming conventions for the huIFN-alpha sequences are according to AllenG. and Diaz M. O. (1996) J. Interferon and Cytokine Res. 16:181-184.Amino acid residue positions in SEQ ID NOs:45-46 and 48-57 which areidentical to SEQ ID NO:13 are indicated with a period (.), and gaps inthe sequence are indicated with a dash (-).

FIG. 3 shows an alignment of the sequence of a polypeptide of theinvention (SEQ ID NO:38) with human interferon-alpha polypeptidesequences SEQ ID NOs:45-46 and 48-57. Amino acid residue positions inSEQ ID NOs:45-46 and 48-57 which are identical to SEQ ID NO:38 areindicated with a period (.), and gaps in the sequence are indicated witha dash (-).

FIG. 4 shows the BLOSUM62 substitution matrix.

FIGS. 5A, 5B and 5C show examples of calculations of alignment scoresused to determine optimal sequence alignments, using the followingparameters: BLOSUM62 matrix, gap open penalty=11, and gap extensionpenalty=1.

FIGS. 6 and 7 show E. coli expression vectors designatedOripBR-URA3-lacI_(—)14epi18-mut2 and OripBR-URA3-lacI_(—)25epi19-mut,respectively.

FIG. 8 shows the separation via cation exchange HPLC of positionalisomers of an exemplary PEGylated interferon-alpha conjugate.

FIG. 9 shows an SDS_PAGE gel of fractions from the cation exchange HPLCseparation of FIG. 8.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless otherwise defined herein or in the remainder of thespecification, all technical and scientific terms used herein have thesame meaning as commonly understood by those of ordinary skill in theart to which the invention belongs.

A “polypeptide sequence” (e.g., a protein, polypeptide, peptide, etc.)is a polymer of amino acids comprising naturally occurring amino acidsor artificial amino acid analogues, or a character string representingan amino acid polymer, depending on context. Given the degeneracy of thegenetic code, one or more nucleic acids, or the complementary nucleicacids thereof, that encode a specific polypeptide sequence can bedetermined from the polypeptide sequence.

A “polynucleotide sequence” (e.g., a nucleic acid, polynucleotide,oligonucleotide, etc.) is a polymer of nucleotides comprisingnucleotides A, C, T, U, G, or other naturally occurring nucleotides orartificial nucleotide analogues, or a character string representing anucleic acid, depending on context. Either the given nucleic acid or thecomplementary nucleic acid can be determined from any specifiedpolynucleotide sequence.

Numbering of a given amino acid polymer or nucleic acid polymer“corresponds to” or is “relative to” the numbering of a selected aminoacid polymer or nucleic acid polymer when the position of any givenpolymer component (e.g., amino acid, nucleotide, also referred togenerically as a “residue”) is designated by reference to the same or anequivalent position in the selected amino acid or nucleic acid polymer,rather than by the actual numerical position of the component in thegiven polymer. Thus, for example, the numbering of a given amino acidposition in a given polypeptide sequence corresponds to the same orequivalent amino acid position in a selected polypeptide sequence usedas a reference sequence.

An “equivalent position” (for example, an “equivalent amino acidposition” or “equivalent nucleic acid position” or “equivalent residueposition”) is defined herein as a position (such as, an amino acidposition or nucleic acid position or residue position) of a testpolypeptide (or test polynucleotide) sequence which aligns with acorresponding position of a reference polypeptide (or referencepolynucleotide) sequence, when optimally aligned using an alignmentalgorithm as described herein. The equivalent amino acid position of thetest polypeptide need not have the same numerical position number as thecorresponding position of the reference polypeptide; likewise, theequivalent nucleic acid position of the test polynucleotide need nothave the same numerical position number as the corresponding position ofthe reference polynucleotide. As an example, FIG. 2 shows the sequenceof a polypeptide of the invention (SEQ ID NO:13) optimally aligned withvarious known human interferon-alpha polypeptide sequences. In thisexample, amino acid position number 48 of SEQ ID NO:13 is considered tobe an equivalent amino acid position to (i.e. is “equivalent to”) thatof amino acid position number 47 of SEQ ID NO:46 (huIFN-alpha 2b), sinceamino acid number 48 of SEQ ID NO:13 aligns with amino acid number 47 ofSEQ ID NO:46. In other words, amino acid position 48 of SEQ ID NO:13“corresponds to” amino acid position 47 of SEQ ID NO:46. Likewise,residue A48 (Ala48) in SEQ ID NO:13 is understood to correspond toresidue F48 (Phe48) in, e.g., SEQ ID NO:1, so that for example thesubstitution A48L relative to SEQ ID NO:13 is understood to correspondto the substitution F48L relative to, e.g., SEQ ID NO:1 (and so on).

Two polypeptide sequences are “optimally aligned” when they are alignedusing defined parameters, i.e., a defined amino acid substitutionmatrix, gap existence penalty (also termed gap open penalty), and gapextension penalty, so as to arrive at the highest similarity scorepossible for that pair of sequences. The BLOSUM62 matrix (Henikoff andHenikoff (1992) Proc. Natl. Acad. Sci. USA 89(22):10915-10919) is oftenused as a default scoring substitution matrix in polypeptide sequencealignment algorithms (such as BLASTP). The gap existence penalty isimposed for the introduction of a single amino acid gap in one of thealigned sequences, and the gap extension penalty is imposed for eachresidue position in the gap. Unless otherwise stated, alignmentparameters employed herein are: BLOSUM62 scoring matrix, gap existencepenalty=11, and gap extension penalty=1. The alignment score is definedby the amino acid positions of each sequence at which the alignmentbegins and ends (e.g. the alignment window), and optionally by theinsertion of a gap or multiple gaps into one or both sequences, so as toarrive at the highest possible similarity score, as described in moredetail below in the section entitled “Percent Sequence Identity”.

The terminology used for identifying amino acid positions and amino acidsubstitutions is illustrated as follows: F48 indicates position number48 is occupied by a phenylalanine (Phe) residue in a reference aminoacid sequence, such as SEQ ID NO:1. F48A indicates that thephenylalanine residue of position 48 has been substituted with a alanine(Ala) residue. Alternative substitutions are indicated with a “/”, e.g.,F48A/L means an amino acid sequence in which the phenylalanine residueat position 48 is substituted with an alanine or a leucine residue.Multiple substitutions may sometimes be indicated with a “+”, e.g.H47Q+F48A/L means an amino acid sequence which comprises a substitutionof the histidine residue at position 47 with an glutamine residue and asubstitution of the phenylalanine residue in position 48 with an alanineor a leucine residue Deletions are indicated by an asterix. For example,H47* indicates that the histidine residue in position 47 has beendeleted. Deletions of two or more continuous amino acids may beindicated as follows, e.g., R161*-E166* indicates the deletion ofresidues R161-E166 inclusive (that is, residues 161, 162, 163, 164, 164,and 166 are deleted). Insertions are indicated the following way:Insertion of an additional serine residue after the histidine residuelocated at position 47 is indicated as H47HS. Combined substitutions andinsertions are indicated in the following way: Substitution of thehistidine residue at position 47 with a serine residue and insertion ofan alanine residue after the position 47 amino acid residue is indicatedas H47SA.

Unless otherwise indicated, the position numbering of amino acidresidues recited herein is relative to the amino acid sequence SEQ IDNO:1. It is to be understood that while the examples and modificationsto the parent polypeptide are generally provided herein relative to thesequence SEQ ID NO:1 (or relative to another specified sequence), theexamples pertain to other polypeptides of the invention, and themodifications described herein may be made in equivalent amino acidpositions of any of the other polypeptides described herein. Thus, as anexample, the substitution F48L relative to SEQ ID NO:1 is understood tocorrespond to the substitution A48L in SEQ ID NO:13, and so on.

The term “exhibiting (e.g., exhibits, or having, or has) aninterferon-alpha activity” is intended to indicate that the polypeptideor conjugate of the invention has at least one activity exhibited by areference interferon-alpha polypeptide (such as, for example, a humaninterferon-alpha polypeptide, e.g., huIFN-alpha 2b identified herein asSEQ ID NO:46, huIFN-alpha 2a identified herein as SEQ ID NO:47,hIFN-alpha 8b identified herein as SEQ ID NO:51, or any other interferonalpha polypeptide known in the art, such as, for example, those listedin Allen G. and Diaz M. O. (1996, supra)). Such activity includes theability to signal through an interferon-alpha receptor, as evidenced by,for example, one or more of: inhibition of viral replication invirus-infected cells (“antiviral activity”); enhancement ofdifferentiation of naïve T-cells to a T_(H)1 phenotype and/orsuppression of differentiation of naïve T-cells to a T_(H)2 phenotype(“T_(H)1 differentiation activity”); or inhibition of cell proliferation(“antiproliferative activity”). The one or more interferon-alphaactivity is assayed using assays known in the art and/or described inthe Examples.

A polypeptide or a conjugate exhibiting an interferon-alpha activity isconsidered to have such activity when it displays a measurable activity,e.g., a measurable antiviral activity, antiproliferative activity, orT_(H)1 differentiation activity (e.g., as determined by assays known inthe art and/or described in the Examples). One of skill in the artrecognizes that what constitutes a measurable activity depends in parton the nature of the assay being undertaken, but as a general guidelinea measurable activity is one in which the assay signal generated in thepresence of the test compound (e.g., a polypeptide of the invention) isquantifiably different than the assay signal generated in the absence ofthe test compound. It is to be understood that a polypeptide orconjugate of the invention need not exhibit all of the known activitiesof a particular reference interferon-alpha, or exhibit such activitiesto the same extent as the reference interferon-alpha. In some instancesthe activity exhibited by a polypeptide or conjugate of the invention(as evidenced, e.g., by an EC₅₀, specific activity, or other valuerelated to activity) may be about equal to, be less than, or be greaterthan that of the particular activity exhibited by the referenceinterferon-alpha.

A “variant” is a polypeptide comprising a sequence which differs in oneor more amino acid position(s) from that of a parent polypeptidesequence. For example, a variant may comprise a sequence which differsfrom the parent polypeptides sequence in up to 10% of the total numberof residues of the parent polypeptide sequence, such as in up to 8% ofthe residues, e.g., in up to 6%, 5%, 4%, 3% 2% or 1% of the total numberof residue of the parent polypeptide sequence. For example, a variant ofSEQ ID NO:1 may comprise a sequence which differs from SEQ ID NO:1 in1-16 amino acid positions (such as in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or 16 amino acid positions), e.g. in 1-15 amino acidpositions, in 1-14 amino acid positions, in 1-13 amino acid positions,in 1-12 amino acid positions, in 1-11 amino acid positions, in 1-10amino acid positions, in 1-9 amino acid positions, in 1-8 amino acidpositions, in 1-7 amino acid positions, in 1-6 amino acid positions, in1-5 amino acid positions, in 1-4 amino acid positions, in 1-3 amino acidpositions, or in 1-2 amino acid positions.

The term “parent polypeptide” or “parent interferon-alpha” is intendedto indicate the polypeptide sequence to be modified in accordance withthe present invention. The parent polypeptide sequence may be that of anaturally occurring IFN-alpha (such as a mammalian IFN-alpha, e.g., aprimate IFN-alpha, such as a human IFN-alpha, such as a huIFN-alphapolypeptide identified herein as SEQ ID NOs:45-57, or other huIFN-alphasequence such as those described herein and/or in Allen G. and Diaz M.O. (1996), supra). The parent polypeptide sequence may be that of anon-naturally occurring (i.e., “synthetic”) interferon-alpha, such asIFN-alpha Con1 (SEQ ID NO:58). In some instances, the parent polypeptideto be modified may itself be a polypeptide of the invention, such as,for example, any one of SEQ ID NOs:2-35 and SEQ ID NOs:37-44.

“Naturally occurring” as applied to an object refers to the fact thatthe object can be found in nature as distinct from being artificiallyproduced by man. For example, a polypeptide or polynucleotide sequencethat is present in an organism (including viruses, bacteria, protozoa,insects, plants or mammalian tissue) that can be isolated from a sourcein nature and which has not been intentionally modified by man in thelaboratory is naturally occurring. “Non-naturally occurring” (alsotermed “synthetic” or “artificial”) as applied to an object means thatthe object is not naturally-occurring—i.e., the object cannot be foundin nature as distinct from being artificially produced by man.

A “fragment” or “subsequence” is any portion of an entire sequence, upto but not including the entire sequence. Thus, a fragment orsubsequence refers to a sequence of amino acids or nucleic acids thatcomprises a part of a longer sequence of amino acids (e.g., polypeptide)or nucleic acids (e.g., polynucleotide).

One type of fragment contemplated by the present invention is a fragmentin which amino acid residues are removed from the N-terminus or theC-terminus of the parent polypeptide (or both); such a polypeptide isconsidered to be “N-terminally truncated” or “C-terminally truncated”,respectively. It is known that deletion of at least the first four aminoacids from the N-terminus does not significantly affect interferon-alphaactivity (Lydon, N. B. et al. (1985) Biochemistry 24: 4131-41).Furthermore, variants retaining interferon-alpha activity have beendescribed wherein between 7 and 11 amino acids have been deleted fromthe C-terminus (Cheetham B. F. et al. (1991) Antiviral Res. 15(1):27-39;Chang N. T. et al. (1983) Arch. Biochem Biophys. 221(2): 585-589; FrankeA. E. et al. (1982) DNA 1(3):223-230).

A “receptor” e.g., an “interferon-alpha receptor” (also known as a “TypeI interferon receptor”) is a receptor which is activated in cells by aninterferon-alpha, e.g., binds an interferon-alpha and initiatesintracellular signaling, such as a type I interferon receptor comprisingreceptor subunits IFNAR-2 and IFNAR-1 (Domanski et al. (1998) J. Biol.Chem. 273(6):3144-3147; Mogensen et al., (1999) Journal of Interferonand Cytokine Research, 19:1069-1098). In the context of this invention,receptor is also meant to include truncated forms of a full-lengthreceptor molecule, such as for example a receptor molecule which lacks amembrane-binding portion, such as a soluble form of a receptor molecule(also known as a “soluble receptor”) which comprises an extracelluarbinding domain, which binds an interferon-alpha, but may not necessarilybind to a membrane and/or initiate intracellular signaling.

A “specific binding affinity” between two molecules, e.g., a ligand anda receptor, means a preferential binding of one molecule for another ina mixture of molecules. The binding of the molecules is typicallyconsidered specific if the binding affinity is about 1×10⁴ M⁻¹ to about1×10⁹ M⁻¹ or greater (i.e., K_(D) of about 10⁻⁴ to 10⁻⁹ M or less).Binding affinity of a ligand and a receptor may be measured by standardtechniques known to those of skill in the art. Non-limiting examples ofwell-known techniques for measuring binding affinities include Biacore®technology (Biacore AB, Sweden), isothermal titration microcalorimetry(MicroCal LLC, Northampton, Mass. USA), ELISA, and FACS. For example,FACS or other sorting methods may be used to select for populations ofmolecules (such as for example, cell surface-displayed ligands) whichspecifically bind to the associated binding pair member (such as areceptor, e.g., a soluble receptor). Ligand-receptor complexes may bedetected and sorted e.g., by fluorescence (e.g., by reacting the complexwith a fluorescent antibody that recognizes the complex). Molecules ofinterest which bind an associated binding pair member (e.g., receptor)are pooled and re-sorted in the presence of lower concentrations ofreceptor. By performing multiple rounds sorting in the presence ofdecreasing concentrations of receptor (an exemplary concentration rangebeing on the order of 10⁻⁶ M down to 10⁻⁹ M, i.e., 1 micromolar (μM)down to 1 nanomolar (nM), or less, depending on the nature of theligand-receptor interaction), populations of the molecule of interestexhibiting specific binding affinity for the receptor may be isolated.

A polypeptide, nucleic acid, or other component is “isolated” when it ispartially or completely separated from components with which it isnormally associated (other peptides, polypeptides, proteins (includingcomplexes, e.g., polymerases and ribosomes which may accompany a nativesequence), nucleic acids, cells, synthetic reagents, cellularcontaminants, cellular components, etc.), e.g., such as from othercomponents with which it is normally associated in the cell from whichit was originally derived. A polypeptide, nucleic acid, or othercomponent is isolated when it is partially or completely recovered orseparated from other components of its natural environment such that itis the predominant species present in a composition, mixture, orcollection of components (i.e., on a molar basis it is more abundantthan any other individual species in the composition). In someinstances, the preparation consists of more than about 60%, 70% or 75%,typically more than about 80%, or preferably more than about 90% of theisolated species.

A “substantially pure” or “isolated” nucleic acid (e.g., RNA or DNA),polypeptide, protein, or composition also means where the object species(e.g., nucleic acid or polypeptide) comprises at least about 50, 60, or70 percent by weight (on a molar basis) of all macromolecular speciespresent. A substantially pure or isolated composition can also compriseat least about 80, 90, or 95 percent by weight of all macromolecularspecies present in the composition. An isolated object species can alsobe purified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of derivatives of a singlemacromolecular species. The term “purified” generally denotes that anucleic acid, polypeptide, or protein gives rise to essentially one bandin an electrophoretic gel. It typically means that the nucleic acid,polypeptide, or protein is at least about 50% pure, 60% pure, 70% pure,75% pure, more preferably at least about 85% pure, and most preferablyat least about 99% pure.

The term “isolated nucleic acid” may refer to a nucleic acid (e.g., DNAor RNA) that is not immediately contiguous with both of the codingsequences with which it is immediately contiguous (i.e., one at the 5′and one at the 3′ end) in the naturally occurring genome of the organismfrom which the nucleic acid of the invention is derived. Thus, this termincludes, e.g., a cDNA or a genomic DNA fragment produced by polymerasechain reaction (PCR) or restriction endonuclease treatment, whether suchcDNA or genomic DNA fragment is incorporated into a vector, integratedinto the genome of the same or a different species than the organism,including, e.g., a virus, from which it was originally derived, linkedto an additional coding sequence to form a hybrid gene encoding achimeric polypeptide, or independent of any other DNA sequences. The DNAmay be double-stranded or single-stranded, sense or antisense.

A “recombinant polynucleotide” or a “recombinant polypeptide” is anon-naturally occurring polynucleotide or polypeptide which may includenucleic acid or amino acid sequences, respectively, from more than onesource nucleic acid or polypeptide, which source nucleic acid orpolypeptide can be a naturally occurring nucleic acid or polypeptide, orcan itself have been subjected to mutagenesis or other type ofmodification. A nucleic acid or polypeptide may be deemed “recombinant”when it is synthetic or artificial or engineered, or derived from asynthetic or artificial or engineered polypeptide or nucleic acid. Arecombinant nucleic acid (e.g., DNA or RNA) can be made by thecombination (e.g., artificial combination) of at least two segments ofsequence that are not typically included together, not typicallyassociated with one another, or are otherwise typically separated fromone another. A recombinant nucleic acid can comprise a nucleic acidmolecule formed by the joining together or combination of nucleic acidsegments from different sources and/or artificially synthesized. A“recombinant polypeptide” often refers to a polypeptide that resultsfrom a cloned or recombinant nucleic acid. The source polynucleotides orpolypeptides from which the different nucleic acid or amino acidsequences are derived are sometimes homologous (i.e., have, or encode apolypeptide that encodes, the same or a similar structure and/orfunction), and are often from different isolates, serotypes, strains,species, of organism or from different disease states, for example.

The term “recombinant” when used with reference, e.g., to a cell,polynucleotide, vector, protein, or polypeptide typically indicates thatthe cell, polynucleotide, or vector has been modified by theintroduction of a heterologous (or foreign) nucleic acid or thealteration of a native nucleic acid, or that the protein or polypeptidehas been modified by the introduction of a heterologous amino acid, orthat the cell is derived from a cell so modified. Recombinant cellsexpress nucleic acid sequences that are not found in the native(non-recombinant) form of the cell or express native nucleic acidsequences that would otherwise be abnormally expressed, under-expressed,or not expressed at all. The term “recombinant” when used with referenceto a cell indicates that the cell replicates a heterologous nucleicacid, or expresses a polypeptide encoded by a heterologous nucleic acid.Recombinant cells can contain coding sequences that are not found withinthe native (non-recombinant) form of the cell. Recombinant cells canalso contain coding sequences found in the native form of the cellwherein the coding sequences are modified and re-introduced into thecell by artificial means. The term also encompasses cells that contain anucleic acid endogenous to the cell that has been modified withoutremoving the nucleic acid from the cell; such modifications includethose obtained by gene replacement, site-specific mutation,recombination, and related techniques.

The term “recombinantly produced” refers to an artificial combinationusually accomplished by either chemical synthesis means, recursivesequence recombination of nucleic acid segments or other diversitygeneration methods (such as, e.g., shuffling) of nucleotides, ormanipulation of isolated segments of nucleic acids, e.g., by geneticengineering techniques known to those of ordinary skill in the art.“Recombinantly expressed” typically refers to techniques for theproduction of a recombinant nucleic acid in vitro and transfer of therecombinant nucleic acid into cells in vivo, in vitro, or ex vivo whereit may be expressed or propagated.

A “recombinant expression cassette” or simply an “expression cassette”is a nucleic acid construct, generated recombinantly or synthetically,with nucleic acid elements that are capable of effecting expression of astructural gene in hosts compatible with such sequences. Expressioncassettes include at least promoters and optionally, transcriptiontermination signals. Typically, the recombinant expression cassetteincludes a nucleic acid to be transcribed (e.g., a nucleic acid encodinga desired polypeptide), and a promoter. Additional factors necessary orhelpful in effecting expression may also be used as described herein.For example, an expression cassette can also include nucleotidesequences that encode a signal sequence that directs secretion of anexpressed protein from the host cell. Transcription termination signals,enhancers, and other nucleic acid sequences that influence geneexpression, can also be included in an expression cassette.

An “immunogen” refers to a substance capable of provoking an immuneresponse, and includes, e.g., antigens, autoantigens that play a role ininduction of autoimmune diseases, and tumor-associated antigensexpressed on cancer cells. An immune response generally refers to thedevelopment of a cellular or antibody-mediated response to an agent,such as an antigen or fragment thereof or nucleic acid encoding suchagent. In some instances, such a response comprises a production of atleast one or a combination of CTLs, B cells, or various classes of Tcells that are directed specifically to antigen-presenting cellsexpressing the antigen of interest.

An “antigen” refers to a substance that is capable of eliciting theformation of antibodies in a host or generating a specific population oflymphocytes reactive with that substance. Antigens are typicallymacromolecules (e.g., proteins and polysaccharides) that are foreign tothe host.

An “adjuvant” refers to a substance that enhances an antigen'simmune-stimulating properties or the pharmacological effect(s) of adrug. An adjuvant may non-specifically enhance the immune response to anantigen. “Freund's Complete Adjuvant,” for example, is an emulsion ofoil and water containing an immunogen, an emulsifying agent andmycobacteria. Another example, “Freund's incomplete adjuvant,” is thesame, but without mycobacteria.

A vector is a component or composition for facilitating celltransduction or transfection by a selected nucleic acid, or expressionof the nucleic acid in the cell. Vectors include, e.g., plasmids,cosmids, viruses, YACs, bacteria, poly-lysine, etc. An “expressionvector” is a nucleic acid construct or sequence, generated recombinantlyor synthetically, with a series of specific nucleic acid elements thatpermit transcription of a particular nucleic acid in a host cell. Theexpression vector can be part of a plasmid, virus, or nucleic acidfragment. The expression vector typically includes a nucleic acid to betranscribed operably linked to a promoter. The nucleic acid to betranscribed is typically under the direction or control of the promoter.“Substantially the entire length of a polynucleotide sequence” or“substantially the entire length of a polypeptide sequence” refers to atleast 50%, generally at least about 60%, 70%, or 75%, usually at leastabout 80%, or typically at least about 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more of a length of a polynucleotide sequenceor polypeptide sequence.

The term “immunoassay” includes an assay that uses an antibody orimmunogen to bind or specifically bind an antigen. The immunoassay istypically characterized by the use of specific binding properties of aparticular antibody to isolate, target, and/or quantify the antigen.

The term “subject” as used herein includes, but is not limited to, anorganism; a mammal, including, e.g., a human, non-human primate (e.g.,baboon, orangutan, monkey), mouse, pig, cow, goat, cat, rabbit, rat,guinea pig, hamster, horse, monkey, sheep, or other non-human mammal; anon-mammal, including, e.g., a non-mammalian vertebrate, such as a bird(e.g., a chicken or duck) or a fish, and a non-mammalian invertebrate.

The term “pharmaceutical composition” means a composition suitable forpharmaceutical use in a subject, including an animal or human. Apharmaceutical composition generally comprises an effective amount of anactive agent and a carrier, including, e.g., a pharmaceuticallyacceptable carrier.

The term “effective amount” means a dosage or amount sufficient toproduce a desired result. The desired result may comprise an objectiveor subjective improvement in the recipient of the dosage or amount.

A “prophylactic treatment” is a treatment administered to a subject whodoes not display signs or symptoms of a disease, pathology, or medicaldisorder, or displays only early signs or symptoms of a disease,pathology, or disorder, such that treatment is administered for thepurpose of diminishing, preventing, or decreasing the risk of developingthe disease, pathology, or medical disorder. A prophylactic treatmentfunctions as a preventative treatment against a disease or disorder. A“prophylactic activity” is an activity of an agent, such as a nucleicacid, vector, gene, polypeptide, protein, substance, or compositionthereof that, when administered to a subject who does not display signsor symptoms of pathology, disease or disorder, or who displays onlyearly signs or symptoms of pathology, disease, or disorder, diminishes,prevents, or decreases the risk of the subject developing a pathology,disease, or disorder. A “prophylactically useful” agent or compound(e.g., nucleic acid or polypeptide) refers to an agent or compound thatis useful in diminishing, preventing, treating, or decreasingdevelopment of pathology, disease or disorder.

A “therapeutic treatment” is a treatment administered to a subject whodisplays symptoms or signs of pathology, disease, or disorder, in whichtreatment is administered to the subject for the purpose of diminishingor eliminating those signs or symptoms of pathology, disease, ordisorder. A “therapeutic activity” is an activity of an agent, such as anucleic acid, vector, gene, polypeptide, protein, substance, orcomposition thereof, that eliminates or diminishes signs or symptoms ofpathology, disease or disorder, when administered to a subject sufferingfrom such signs or symptoms. A “therapeutically useful” agent orcompound (e.g., nucleic acid or polypeptide) indicates that an agent orcompound is useful in diminishing, treating, or eliminating such signsor symptoms of a pathology, disease or disorder.

The term “gene” broadly refers to any segment of DNA associated with abiological function. Genes include coding sequences and/or regulatorysequences required for their expression. Genes also includenon-expressed DNA nucleic acid segments that, e.g., form recognitionsequences for other proteins (e.g., promoter, enhancer, or otherregulatory regions). Genes can be obtained from a variety of sources,including cloning from a source of interest or synthesizing from knownor predicted sequence information, and may include sequences designed tohave desired parameters.

Generally, the nomenclature used hereafter and the laboratory proceduresin cell culture, molecular genetics, molecular biology, nucleic acidchemistry, and protein chemistry described below are those well knownand commonly employed by those of ordinary skill in the art. Standardtechniques, such as described in Sambrook et al., Molecular Cloning—ALaboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1989 (hereinafter “Sambrook”) and CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc. (1994, supplemented through 1999)(hereinafter “Ausubel”), are used for recombinant nucleic acid methods,nucleic acid synthesis, cell culture methods, and transgeneincorporation, e.g., electroporation, injection, gene gun, impressingthrough the skin, and lipofection. Generally, oligonucleotide synthesisand purification steps are performed according to specifications. Thetechniques and procedures are generally performed according toconventional methods in the art and various general references which areprovided throughout this document. The procedures therein are believedto be well known to those of ordinary skill in the art and are providedfor the convenience of the reader.

As used herein, an “antibody” refers to a protein comprising one or morepolypeptides substantially or partially encoded by immunoglobulin genesor fragments of immunoglobulin genes. The term antibody is used to meanwhole antibodies and binding fragments thereof. The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as myriad immunoglobulinvariable region genes. Light chains are classified as either kappa orlambda. Heavy chains are classified as gamma, mu, alpha, delta, orepsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA,IgD and IgE, respectively. A typical immunoglobulin (e.g., antibody)structural unit comprises a tetramer. Each tetramer is composed of twoidentical pairs of polypeptide chains, each pair having one “light”(about 25 KDa) and one “heavy” chain (about 50-70 KDa). The N-terminusof each chain defines a variable region of about 100 to 110 or moreamino acids primarily responsible for antigen recognition. The termsvariable light chain (VL) and variable heavy chain (VH) refer to theselight and heavy chains, respectively.

Antibodies also include single-armed composite monoclonal antibodies,single chain antibodies, including single chain Fv (sFv) antibodies inwhich a variable heavy and a variable light chain are joined together(directly or through a peptide linker) to form a continuous polypeptide,as well as diabodies, tribodies, and tetrabodies (Pack et al. (1995) JMol Biol 246:28; Biotechnol 11:1271; and Biochemistry 31:1579). Theantibodies are, e.g., polyclonal, monoclonal, chimeric, humanized,single chain, Fab fragments, fragments produced by an Fab expressionlibrary, or the like.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents.

An “antigen-binding fragment” of an antibody is a peptide or polypeptidefragment of the antibody that binds an antigen. An antigen-binding siteis formed by those amino acids of the antibody that contribute to, areinvolved in, or affect the binding of the antigen. See Scott, T. A. andMercer, E. I., Concise Encyclopedia: Biochemistry and Molecular Biology(de Gruyter, 3d ed. 1997), and Watson, J. D. et al., Recombinant DNA (2ded. 1992) (hereinafter “Watson, Recombinant DNA”), each of which isincorporated herein by reference in its entirety for all purposes.

The term “screening” describes, in general, a process that identifiesoptimal molecules of the present invention, such as, e.g., polypeptidesof the invention, and related fusion polypeptides including the same,and nucleic acids encoding all such molecules. Several properties ofthese respective molecules can be used in selection and screening, forexample: an ability of a respective molecule to bind a ligand or to areceptor, to inhibit cell proliferation, to inhibit viral replication invirus-infected cells, to induce or inhibit cellular cytokine production,to alter an immune response, e.g., induce or inhibit a desired immuneresponse, in a test system or an in vitro, ex vivo or in vivoapplication. In the case of antigens, several properties of the antigencan be used in selection and screening including antigen expression,folding, stability, immunogenicity and presence of epitopes from severalrelated antigens.

“Selection” is a form of screening in which identification and physicalseparation are achieved simultaneously by, e.g., expression of aselection marker, which, in some genetic circumstances, allows cellsexpressing the marker to survive while other cells die (or vice versa).Screening markers include, for example, luciferase, beta-galactosidaseand green fluorescent protein, and the like. Selection markers includedrug and toxin resistance genes, and the like. Another mode of selectioninvolves physical sorting based on a detectable event, such as bindingof a ligand to a receptor, reaction of a substrate with an enzyme, orany other physical process which can generate a detectable signal eitherdirectly (e.g., by utilizing a chromogenic substrate or ligand) orindirectly (e.g., by reacting with a chromogenic secondary antibody).Selection by physical sorting can by accomplished by a variety ofmethods, such as by FACS in whole cell or microdroplet formats.

An “exogenous nucleic acid,” “exogenous DNA segment,” “heterologoussequence,” or “heterologous nucleic acid,” as used herein, is one thatoriginates from a source foreign to the particular host cell, or, iffrom the same source, is modified from its original form. Thus, aheterologous gene in a host cell includes a gene that is endogenous tothe particular host cell, but has been modified. Modification of aheterologous sequence in the applications described herein typicallyoccurs through the use of recursive sequence recombination. The termsrefer to a DNA segment which is foreign or heterologous to the cell, orhomologous to the cell but in a position within the host cell nucleicacid in which the element is not ordinarily found. Exogenous DNAsegments are expressed to yield exogenous polypeptides.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences and as wellas the sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al. (1991) NucleicAcid Res 19:5081; Ohtsuka et al. (1985) J Biol Chem 260:2605-2608;Cassol et al. (1992); Rossolini et al. (1994) Mol Cell Probes 8:91-98).The term nucleic acid is used interchangeably with gene, cDNA, and mRNAencoded by a gene.

“Nucleic acid derived from a gene” refers to a nucleic acid for whosesynthesis the gene, or a subsequence thereof, has ultimately served as atemplate. Thus, an mRNA, a cDNA reverse transcribed from an mRNA, an RNAtranscribed from that cDNA, a DNA amplified from the cDNA, an RNAtranscribed from the amplified DNA, etc., are all derived from the geneand detection of such derived products is indicative of the presenceand/or abundance of the original gene and/or gene transcript in asample.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itincreases the transcription of the coding sequence. Operably linkedmeans that the DNA sequences being linked are typically contiguous and,where necessary to join two protein coding regions, contiguous and inreading frame. However, since enhancers generally function whenseparated from the promoter by several kilobases and intronic sequencesmay be of variable lengths, some polynucleotide elements may be operablylinked but not contiguous.

The term “cytokine” includes, for example, interleukins, interferons,chemokines, hematopoietic growth factors, tumor necrosis factors andtransforming growth factors. In general these are low molecular weightproteins that regulate maturation, activation, proliferation, anddifferentiation of cells of the immune system.

In the present description and claims, any reference to “a” component,e.g. in the context of a non-polypeptide moiety, an amino acid residue,a substitution, a buffer, a cation, etc., is intended to refer to one ormore of such components, unless stated otherwise or unless it is clearfrom the particular context that this is not the case. For example, theexpression “a component selected from the group consisting of A, B andC” is intended to include all combinations of A, B and C, e.g., A, B, C,A+B, A+C, B+C or A+B+C.

Various additional terms are defined or otherwise characterized herein.

Molecules and Methods of the Invention

Molecules of the invention (e.g., polypeptides of the invention,conjugates of the invention, and nucleic acids encoding saidpolypeptides) are useful for the treatment of diseases and disorderswhich are responsive to treatment by interferon-alpha, particularlydiseases associated with viral infection, such as, for example,infection by HCV.

Patients with chronic HCV infection have viral loads typically in therange of 10⁴-10⁷ copies of HCV RNA/ml of serum prior to treatment. Upontreatment with IFN-alpha, viral load in these patientscharacteristically undergoes two distinct log-linear phases of decline(FIG. 1B; Neumann A. U., et al. (1998) Science 282:103-107). The initialrapid drop in viral load that occurs within the first two days ofIFN-alpha therapy is believed to be due to interferon-alpha mediatedreduction in virus production in the infected liver cells andconcomitant protection of naïve cells against infection. The rate ofviral production reaches a new steady state at about two days, at whichtime a second less rapid log-linear phase of viral clearance isobserved. This second phase of viral clearance is generally believed tobe due in part to T-cell mediated killing of infected liver cells(Neumann, et al., supra). IFN-alpha is believed to play a key role inthis biological response through the stimulation of antigen specific Tcells to differentiate into T_(H)1 cells. Furthermore, the mode ofaction of Ribavirin is believed to be due to augmentation of the T_(H)1response, and is thought to be the mechanistic basis of its efficacy incombination therapy with IFN-alpha. HCV-infected patients who arenon-responsive to interferon-alpha therapies currently in use (generallytermed “non-responders”) exhibit much shallower viral load clearanceprofiles (FIG. 1A).

Although the present invention is not intended to be limited by aparticular theory of underlying mechanism, it is proposed that antiviralactivity in surrogate assay systems (such as those described in moredetail herein) may be predictive of interferon-alpha efficacy, in forexample the first phase of viral clearance. Exemplary antiviral assays,described in the Examples section, monitor the effectiveness ofIFN-alpha in protecting against the cytopathic effect ofEncephalomyocarditis Virus (EMCV) in HuH7 human liver-derived cells as asurrogate system for effectiveness against HCV in human liver cells.Other useful virus/cell assay systems include EMCV in WISH humanamnion-derived cells, EMCV in HeLa human cervical carcinoma cells,Vesicular Stomatitis Virus (VSV) in HuH7 cells, Vaccinia Virus (VV) inHeLa cells, Yellow Fever Virus (YFV) in HepG2 human hepatocarcinomacells, as well as Human Immunodeficiency Virus (HIV) in primary CD4+T-cells. Example 2 shows antiviral activities of representativepolypeptides of the invention in the EMCV/HuH7 and EMCV/HeLa antiviralactivity assays.

Other surrogate assay systems useful for monitoring effectivenessagainst HCV in infected hepatocytes include HCV replicon systems, asdescribed, for example, by Lohmann V., et al., (1999) Science285(5424):285-293; Randall G. and Rice C. M. (2001) Curr Opin Infect Dis14(6):743-7477; and Bartenschlager, R. (2002) Nature Reviews/DrugDiscovery 1:911. An example of a useful in vivo host system formonitoring HCV antiviral efficacy is a chimeric human liver SCID mouse,as described by Mercer, et al. (2001) Nature Medicine 7(8):927-933.

It is furthermore proposed, without being limited by theory, thatenhancement of T_(H)1 differentiation and/or suppression of T_(H)2differentiation by IFN-alpha may be a contributing factor tointerferon-alpha efficacy, for example, in the second phase of viralclearance. According to this theory, evolved IFN-alphas with increasedpotency in these biological activities (i.e., enhancement of T_(H)1differentiation and/or suppression of T_(H)2 differentiation) would bepredicted to have increased efficacy relative to, for example, currentlyapproved therapeutic interferon-alpha molecules administered at the samedosage. An exemplary assay, described in the Examples section herein,monitors the enhancement of T_(H)1 differentiation and/or suppression ofT_(H)2 differentiation by IFN-alpha on naïve T_(H)0 cells, by measuringproduction of cytokines associated with the T_(H)1-phenotype (e.g.,IFN-gamma) and/or the T_(H)2-phenotype (e.g., IL-5, IL4) via ELISA orvia intracellular staining and FACS sorting. Example 3 shows T_(H)1differentiation activities of representative polypeptides of theinvention.

The therapeutic efficacy of IFN-alpha molecules tends to be diminishedin part due to dose-limiting toxicities, e.g. thrombocytopenia andneutropenia. Although the present invention is not intended to belimited by a particular theory of underlying mechanism, it is proposedthat such toxicity may be associated with anti-proliferative effects ofIFN-alpha on platelet and neutrophil precursors, and thatantiproliferative activity in surrogate assay systems (such as thosedescribed herein) may be predictive of the relative toxicity of aninterferon-alpha molecule. Thus, dose-limiting toxicities associatedwith IFN-alpha therapy may be diminished in IFN-alpha molecules thatexhibit reduced antiproliferative activity relative to, for example,currently approved therapeutic interferon-alpha molecules, such asROFERON®-A (Interferon alfa-2a, recombinant; Hoffmann-La Roche Inc.),INTRON® A (Interferon alfa-2b, recombinant; Schering Corporation), andINFERGEN® (interferon alfacon-1; InterMune, Inc.). An exemplaryantiproliferative activity assay, described in the Examples sectionherein, monitors the effect of IFN-alpha on the proliferation of humanDaudi lymphoid cells. Alternatively, or in addition, dose-limitingtoxicities may be reduced as a result of administering moretherapeutically active molecules, which would permit dosing in lowerconcentrations or at lower frequency than currently approved molecules.Example 4 shows the Daudi antiproliferative activities of representativepolypeptides of the invention.

It is an object of the invention to provide novel interferon-alphapolypeptides, and nucleic acids which encode the polypeptides.Polypeptides of the invention are useful for the treatment of diseasesand disorders which are responsive to treatment by interferon-alpha,particularly diseases associated with viral infection, such as, forexample, infection by HCV. Some polypeptides of the invention exhibit aninterferon-alpha activity, such as, for example, antiviral activity,antiproliferative activity, and/or T_(H)1 differentiation activity. Somepolypeptides of the invention exhibit one or more of the followingproperties: increased or decreased antiviral activity compared to areference IFN-alpha polypeptide; increased or decreased T_(H)1differentiation activity compared to a reference IFN-alpha polypeptide;increased or decreased antiproliferative activity compared to areference IFN-alpha polypeptide. The reference IFN-alpha polypeptide maycomprise a sequence of a non-naturally occurring interferon-alpha, suchas IFN-alpha Con1 (SEQ ID NO:58), or may comprise a sequence of anaturally-occurring (i.e., wild-type) interferon-alpha polypeptide.Examples of sequences of naturally occurring interferon-alphapolypeptides include sequences of human IFN-alpha polypeptides, such as,for example, huIFN-alpha 2b (SEQ ID NO:46), huIFN-alpha 2a (SEQ IDNO:47), huIFN-alpha 2c (SEQ ID NO:46 with position 34=Arg), huIFN-alpha8b (SEQ ID NO:52), huIFN-alpha 8a (SEQ ID NO:52 with positions 98=Val,99=Leu, 100=Cys, and 101=Asp), huIFN-alpha 8c (SEQ ID NO:52 withposition 161=Asp and amino acids at positions 162-166 deleted),huIFN-alpha 14a (SEQ ID NO:54), huIFN-alpha 14c (SEQ ID NO:54 withposition 152=Leu), or a sequence of any other naturally occurring humaninterferon alpha polypeptide, such as those listed in Allen G. and DiazM. O. (1996), supra.

In another aspect, the invention provides interferon-alpha polypeptideswhich exhibit enhanced efficacy in clearing a virus from virus-infectedcells, compared to a reference interferon-alpha molecule, such as onecurrently employed as a therapeutic (such as, for example, ROFERON-A,INTRON A, or INFERGEN). Exemplary viruses include, but are not limitedto, viruses of the Flaviviridae family, such as, for example, HepatitisC Virus, Yellow Fever Virus, West Nile Virus, Japanese EncephalitisVirus, Dengue Virus, and Bovine Viral Diarrhea Virus; viruses of theHepadnaviridae family, such as, for example, Hepatitis B Virus; virusesof the Picornaviridae family, such as, for example, EncephalomyocarditisVirus, Human Rhinovirus, and Hepatitis A Virus; viruses of theRetroviridae family, such as, for example, Human Immunodeficiency Virus,Simian Immunodeficiency Virus, Human T-Lymphotropic Virus, and RousSarcoma Virus; viruses of the Coronaviridae family, such as, forexample, SARS coronavirus; viruses of the Rhabdoviridae family, such as,for example, Rabies Virus and Vesicular Stomatitis Virus, viruses of theParamyxoviridae family, such as, for example, Respiratory SyncytialVirus and Parainfluenza Virus, viruses of the Papillomaviridae family,such as, for example, Human Papillomavirus, and viruses of theHerpesviridae family, such as, for example, Herpes Simplex Virus. Suchenhanced efficacy may arise from enhanced antiviral activity, enhancedT_(H)1-differentiation activity, or both, relative to the referencemolecule. For example, some interferon-alpha polypeptides of theinvention may be particularly useful in clearing viruses or viralstrains that show poor response to treatment with interferon-alphamolecules currently in use, such as, for example, Genotype 1 of HCV.

Some polypeptides of the invention exhibit an increased ratio of(antiviral activity/antiproliferative activity) compared to thereference IFN-alpha molecule, and/or an increased ratio of (T_(H)1differentiation activity/antiproliferative activity) compared to thereference IFN-alpha molecule. Polypeptides exhibiting such propertiesmay be particularly effective in treatment of viral infections, such as,for example, infection by a virus listed above. Some such polypeptidesmay, for example, provide enhanced therapeutic efficacy overcurrently-approved interferon-alpha molecules in the treatment of HCV,in one or both phases of the biphasic viral clearance profile, and/ormay exhibit reduced toxicity. Some such polypeptides may provideenhanced therapeutic efficacy over currently-approved interferon-alphamolecules in the treatment of Genotype 1 HCV.

It is another object of the invention to provide conjugates, suchconjugates comprising one or more non-polypeptide moiety linked to apolypeptide of the invention, which conjugate exhibits aninterferon-alpha activity (such as one or more of the activities listedabove), and which optionally exhibits other desirable properties, suchas increased serum half-life and/or functional in vivo half-life, and/ordecreased antigenicity, compared to the non-conjugated polypeptide. Somesuch conjugates may exhibit enhanced efficacy in clearing a virus fromcells infected with the virus, compared to a reference interferon-alphamolecule, such as an interferon-alpha conjugate currently employed as atherapeutic (such as, for example, PEGASYS® (Peginterferon alfa-2a;Hoffmann-La Roche, Inc.) or PEG-INTRON® (peginterferon alfa-2b; ScheringCorporation). Exemplary viruses include, but are not limited to, virusesof the Flaviviridae family, such as, for example, Hepatitis C Virus,Yellow Fever Virus, West Nile Virus, Japanese Encephalitis Virus, DengueVirus, and Bovine Viral Diarrhea Virus; viruses of the Hepadnaviridaefamily, such as, for example, Hepatitis B Virus; viruses of thePicornaviridae family, such as, for example, Encephalomyocarditis Virus,Human Rhinovirus, and Hepatitis A Virus; viruses of the Retroviridaefamily, such as, for example, Human Immunodeficiency Virus, SimianImmunodeficiency Virus, Human T-Lymphotropic Virus, and Rous SarcomaVirus; viruses of the Coronaviridae family, such as, for example, SARScoronavirus; viruses of the Rhabdoviridae family, such as, for example,Rabies Virus and Vesicular Stomatitis Virus, viruses of theParamyxoviridae family, such as, for example, Respiratory SyncytialVirus and Parainfluenza Virus, viruses of the Papillomaviridae family,such as, for example, Human Papillomavirus, and viruses of theHerpesviridae family, such as, for example, Herpes Simplex Virus. Suchenhanced efficacy may arise from enhanced antiviral activity, enhancedT_(H)1-differentiation activity, or both, relative to the referencemolecule. For example, some interferon-alpha conjugates of the inventionmay be particularly useful in clearing viruses or viral strains thatshow poor response to treatment with interferon-alpha moleculescurrently in use, such as, for example, Genotype 1 of HCV.

Some conjugates of the invention exhibit an increased ratio of(antiviral activity/antiproliferative activity) compared to thereference IFN-alpha molecule, and/or an increased ratio of (T_(H)1differentiation activity/antiproliferative activity) compared to thereference IFN-alpha molecule. Conjugates exhibiting such properties maybe particularly effective in treatment of viral infections, such asinfection by a virus listed above, such as, for example, HCV. Some suchconjugates may, for example, provide enhanced therapeutic efficacy overcurrently-approved interferon-alpha molecules in the treatment of HCV,in one or both phases of the biphasic viral clearance profile, and/ormay exhibit reduced toxicity. Some such conjugates may provide enhancedtherapeutic efficacy over currently-approved interferon-alpha moleculesin the treatment of Genotype 1 HCV.

It is another object of the invention to provide a method of inhibitingviral replication in virus-infected cells, the method comprisingadministering to the virus-infected cells a polypeptide or conjugate ofthe invention in an amount effective to inhibit viral replication insaid cells. The invention also provides a method of reducing the numberof copies of a virus in virus-infected cells, comprising administeringto the virus-infected cells a polypeptide or conjugate of the inventionin an amount effective to reduce the number of copies of the virus insaid cells. The virus may be a virus of the Flaviviridae family, suchas, for example, Hepatitis C Virus, Yellow Fever Virus, West Nile Virus,Japanese Encephalitis Virus, Dengue Virus, or Bovine Viral DiarrheaVirus; a virus of the Hepadnaviridae family, such as, for example,Hepatitis B Virus; a virus of the Picornaviridae family, such as, forexample, Encephalomyocarditis Virus, Human Rhinovirus, or Hepatitis AVirus; a virus of the Retroviridae family, such as, for example, HumanImmunodeficiency Virus, Simian Immunodeficiency Virus, HumanT-Lymphotropic Virus, or Rous Sarcoma Virus; a virus of theCoronaviridae family, such as, for example, SARS coronavirus; a virus ofthe Rhabdoviridae family, such as, for example, Rabies Virus orVesicular Stomatitis Virus; a virus of the Paramyxoviridae family, suchas, for example, Respiratory Syncytial Virus or Parainfluenza Virus; avirus of the Papillomaviridae family, such as, for example, HumanPapillomavirus; or a virus of the Herpesviridae family, such as, forexample, Herpes Simplex Virus. The virus may for example be an RNAvirus, such as HCV, a DNA virus, such as HBV, or a retrovirus, such asHIV. The cells may be in culture or otherwise isolated from a mammal(i.e., in vitro or ex vivo), or may be in vivo, e.g., in a mammal (e.g.such as a SCID mouse model as described by Mercer, et al. (2001) NatureMedicine. 7(8): 927-933), in a primate, or in man.

The invention also provides a method of enhancing T_(H)1 differentiationof T_(H)0 cells, comprising administering to a population comprisingT_(H)0 cells a polypeptide or conjugate of the invention in an amounteffective to increase the production of a cytokine associated with theT_(H)1-phenotype (e.g., IFN-gamma) and/or decrease the production of acytokine associated with the T_(H)2-phenotype (e.g., IL-4 or IL-5) insaid population. The population may be in culture or otherwise isolatedfrom a mammal (i.e., in vitro or ex vivo), or may be in vivo, e.g., in amammal, in a primate, or in man.

The invention also provides a method of inhibiting proliferation of acell population, comprising contacting the cell population with apolypeptide, variant, or conjugate of the invention in an amounteffective to decrease proliferation of the cell population. The cellpopulation may be in culture or otherwise isolated from a mammal (i.e.,in vitro or ex vivo), or may be in vivo, e.g., in a mammal, a primate,or man.

These and other objects of the invention are discussed in more detailbelow.

Polypeptides of the Invention

The invention provides novel interferon-alpha polypeptides, collectivelyreferred to herein as “polypeptides of the invention”. The term“polypeptide(s) of the invention” is intended throughout to includevariants of the polypeptide sequences disclosed herein. Also included inthis invention are fusion proteins comprising polypeptides of theinvention, and conjugates comprising polypeptides of the invention.

Fragments of various interferon-alpha coding sequences were recursivelyrecombined to form libraries comprising recombinant polynucleotides,from which polypeptides of the invention were derived. Methods forobtaining libraries of recombinant polynucleotides and/or for obtainingdiversity in nucleic acids used as the substrates for recursive sequencerecombination are also described infra.

Exemplary polypeptides of the invention include polypeptides comprisingsequences identified herein as SEQ ID NOs:2-35 and SEQ ID NOs: 37-44(i.e., SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:10, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ IDNO:43, and SEQ ID NO:44). Some such polypeptides further comprise one ormore additional amino acid(s), such as, for example, a methionine addedto the N-terminus. The invention also provides fusion proteins andconjugates comprising these polypeptides, and isolated or recombinantnucleic acids encoding these polypeptides.

The invention also provides isolated or recombinant polypeptides whicheach comprise a sequence which (a) differs in 1 to 16 amino acidpositions from SEQ ID NO:1 and (b) comprises one or more substitution(s)relative to SEQ ID NO:1 selected from the group consisting of F48A/L;V51P; F55A; F65A; F68P; L111A; and V114P. Some such polypeptidescomprise a sequence which differs from SEQ ID NO:1 in 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid positions, e.g., in1-15 amino acid positions, in 1-14 amino acid positions, in 1-13 aminoacid positions, in 1-12 amino acid positions, 1-11 amino acid positions,in 1-10 amino acid positions, in 1-9 amino acid positions, in 1-8 aminoacid positions, in 1-7 amino acid positions, in 1-6 amino acidpositions, in 1-5 amino acid positions, in 1-4 amino acid positions, in1-3 amino acid positions, or in 1-2 amino acid positions. In someinstances, the polypeptide further comprises one or more substitution(s)relative to SEQ ID NO:1 selected from the group consisting of H47Q;V51A; Q52P/E; A53T; F55S; L56V; F57L; Y58H; M61I; N113K; V114E; andE160D. In some instances, the polypeptide exhibits an interferon-alphaactivity (e.g., antiviral activity, T_(H)1 differentiation activity,and/or antiproliferative activity). Some such polypeptides furthercomprise one or more additional amino acid(s), such as a methionineadded to the N-terminus. The invention also provides fusion proteins andconjugates comprising these polypeptides, and isolated or recombinantnucleic acids encoding these polypeptides.

The invention also provides isolated or recombinant polypeptides whicheach comprise a sequence which (a) differs in 1 to 16 amino acidpositions from SEQ ID NO:1 and (b) comprises the substitution F48A/Lrelative to SEQ ID NO:1. Some such polypeptides comprise a sequencewhich differs from SEQ ID NO:1 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, or 16 amino acid positions, e.g., in 1-15 amino acidpositions, in 1-14 amino acid positions, in 1-13 amino acid positions,in 1-12 amino acid positions, 1-11 amino acid positions, in 1-10 aminoacid positions, in 1-9 amino acid positions, in 1-8 amino acidpositions, in 1-7 amino acid positions, in 1-6 amino acid positions, in1-5 amino acid positions, in 1-4 amino acid positions, in 1-3 amino acidpositions, or in 1-2 amino acid positions. Some such polypeptidescomprise the substitution F48A relative to SEQ ID NO:1. In someinstances, the polypeptide further comprises one or more substitution(s)relative to SEQ ID NO:1 selected from the group consisting of H47Q;V51A/P; Q52P/E; A53T; F55A/S; L56V; F65A; F57L; Y58H; M61I; F68P; L111A;N113K; V114E/P; and E160D. In some instances, the polypeptide exhibitsan interferon-alpha activity (e.g., antiviral activity, T_(H)1differentiation activity, and/or antiproliferative activity). Some suchpolypeptides further comprise one or more additional amino acid(s), suchas a methionine added to the N-terminus. The invention also providesfusion proteins and conjugates comprising these polypeptides, andisolated or recombinant nucleic acids encoding these polypeptides.

The invention also provides isolated or recombinant polypeptides whicheach comprise a sequence which (a) differs in 1 to 16 amino acidpositions from SEQ ID NO:1 and (b) comprises the substitution F55Arelative to SEQ ID NO:1. Some such polypeptides comprise a sequencewhich differs from SEQ ID NO:1 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, or 16 amino acid positions, e.g., in 1-15 amino acidpositions, in 1-14 amino acid positions, in 1-13 amino acid positions,in 1-12 amino acid positions, 1-11 amino acid positions, in 1-10 aminoacid positions, in 1-9 amino acid positions, in 1-8 amino acidpositions, in 1-7 amino acid positions, in 1-6 amino acid positions, in1-5 amino acid positions, in 1-4 amino acid positions, in 1-3 amino acidpositions, or in 1-2 amino acid positions. In some instances, thepolypeptide further comprises one or more substitution(s) relative toSEQ ID NO:1 selected from the group consisting of H47Q; F48A/L; V51A/P;Q52P/E; A53T; L56V; F65A; F57L; Y58H; M61I; F68P; L111A; N113K; V114E/P;and E160D. In some instances, the polypeptide exhibits aninterferon-alpha activity (e.g., antiviral activity, T_(H)1differentiation activity, and/or antiproliferative activity). Some suchpolypeptides further comprise one or more additional amino acid(s), suchas a methionine added to the N-terminus. The invention also providesfusion proteins and conjugates comprising these polypeptides, andisolated or recombinant nucleic acids encoding these polypeptides.

The invention also provides isolated or recombinant polypeptides whicheach comprise a sequence which (a) differs in 1 to 16 amino acidpositions from SEQ ID NO:1 and (b) comprises the substitution F65Arelative to SEQ ID NO:1. Some such polypeptides comprise a sequencewhich differs from SEQ ID NO:1 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, or 16 amino acid positions, e.g., in 1-15 amino acidpositions, in 1-14 amino acid positions, in 1-13 amino acid positions,in 1-12 amino acid positions, 1-11 amino acid positions, in 1-10 aminoacid positions, in 1-9 amino acid positions, in 1-8 amino acidpositions, in 1-7 amino acid positions, in 1-6 amino acid positions, in1-5 amino acid positions, in 1-4 amino acid positions, in 1-3 amino acidpositions, or in 1-2 amino acid positions. In some instances, thepolypeptide further comprises one or more substitutions relative to SEQID NO:1 selected from the group consisting of H47Q; F48A/L; V51A/P;Q52P/E; A53T; F55A/S; L56V; F57L; Y58H; M61I; F68P; L111A; N113K;V114E/P; and E160D. In some instances, the polypeptide exhibits aninterferon-alpha activity (e.g., antiviral activity, T_(H)1differentiation activity, and/or antiproliferative activity). Some suchpolypeptides further comprise one or more additional amino acid(s), suchas a methionine added to the N-terminus. The invention also providesfusion proteins and conjugates comprising these polypeptides, andisolated or recombinant nucleic acids encoding these polypeptides.

The invention also provides isolated or recombinant polypeptides whicheach comprise a sequence which (a) differs in 1 to 16 amino acidpositions from SEQ ID NO:1 and (b) comprises the substitution L111Arelative to SEQ ID NO:1. Some such polypeptides comprise a sequencewhich differs from SEQ ID NO:1 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, or 16 amino acid positions, e.g., in 1-15 amino acidpositions, in 1-14 amino acid positions, in 1-13 amino acid positions,in 1-12 amino acid positions, 1-11 amino acid positions, in 1-10 aminoacid positions, in 1-9 amino acid positions, in 1-8 amino acidpositions, in 1-7 amino acid positions, in 1-6 amino acid positions, in1-5 amino acid positions, in 1-4 amino acid positions, in 1-3 amino acidpositions, or in 1-2 amino acid positions. In some instances, thepolypeptide further comprises one or more substitutions relative to SEQID NO:1 selected from the group consisting of H47Q; F48A/L; V51A/P;Q52P/E; A53T; F55A/S; L56V; F65A; F57L; Y58H; M61I; F68P; N113K;V114E/P; and E160D. In some instances, the polypeptide exhibits aninterferon-alpha activity (e.g., antiviral activity, T_(H)1differentiation activity, and/or antiproliferative activity). Some suchpolypeptides further comprise one or more additional amino acid(s), suchas a methionine added to the N-terminus. The invention also providesfusion proteins and conjugates comprising these polypeptides, andisolated or recombinant nucleic acids encoding these polypeptides.

The invention also provides isolated or recombinant polypeptides whicheach comprise a sequence which (a) differs in 0 to 16 amino acidpositions from SEQ ID NO:13 and (b) comprises one or more of: Ala or Leuat position 48; Pro at position 51; Ala at position 55; Ala at position65; Pro at position 68; Ala at position 111; and Pro at position 114(position numbering relative to SEQ ID NO:13). Some such polypeptidescomprise a sequence which differs from SEQ ID NO:13 in 0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid positions, e.g., in0-15 amino acid positions, in 0-14 amino acid positions, in 0-13 aminoacid positions, in 0-12 amino acid positions, in 0-11 amino acidpositions, in 0-10 amino acid positions, in 0-9 amino acid positions, in0-8 amino acid positions, in 0-7 amino acid positions, in 0-6 amino acidpositions, in 0-5 amino acid positions, in 0-4 amino acid positions, in0-3 amino acid positions, in 0-2 amino acid positions, or in 0-1 aminoacid positions. Some such polypeptides comprise an Ala or a Leu atposition 48. Some such polypeptides comprise an Ala at position 48. Insome instances, the polypeptide further comprises one or moresubstitution(s) relative to SEQ ID NO:13 selected from the groupconsisting of H47Q; V51A; Q52P/E; A53T; F55S; L56V; F57L; Y58H; M61I;N113K; V114E; and E160D. In some instances, the polypeptide exhibits aninterferon-alpha activity (e.g., antiviral activity, T_(H)1differentiation activity, and/or antiproliferative activity). Some suchpolypeptides further comprise one or more additional amino acid(s), suchas a methionine added to the N-terminus. The invention also providesfusion proteins and conjugates comprising these polypeptides, andisolated or recombinant nucleic acids encoding these polypeptides.

The invention also provides isolated or recombinant polypeptides whicheach comprise a sequence which (a) differs in 1 to 16 amino acidpositions from SEQ ID NO:36 and (b) comprises one or moresubstitution(s) relative to SEQ ID NO:36 selected from the groupconsisting of M21A; I24P; F48A/L; T51P; S55A; F65A; F68P; F90A; M93P;L111A; V114P; F124A; I127P; and E160D. Some such polypeptides comprise asequence which differs from SEQ ID NO:36 in 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, or 16 amino acid positions, e.g., in 1-15 aminoacid positions, in 1-14 amino acid positions, in 1-13 amino acidpositions, in 1-12 amino acid positions, 1-11 amino acid positions, in1-10 amino acid positions, in 1-9 amino acid positions, in 1-8 aminoacid positions, in 1-7 amino acid positions, in 1-6 amino acidpositions, in 1-5 amino acid positions, in 1-4 amino acid positions, in1-3 amino acid positions, or in 1-2 amino acid positions. In someinstances, the polypeptide further comprises one or more substitution(s)relative to SEQ ID NO:36 selected from the group consisting of P26L;H47Q; T51V; S55P/F; V56L; H58Y; L60M; F90Y; M93L; N95D; N113K; V114E;R125Q; T132K; and L154F. In some instances, the polypeptide exhibits aninterferon-alpha activity (e.g., antiviral activity, T_(H)1differentiation activity, and/or antiproliferative activity). Some suchpolypeptides further comprise one or more additional amino acid(s), suchas a methionine added to the N-terminus. The invention also providesfusion proteins and conjugates comprising these polypeptides, andisolated or recombinant nucleic acids encoding these polypeptides.

The invention also provides isolated or recombinant polypeptides whicheach comprise a sequence which (a) differs in 1 to 16 amino acidpositions from SEQ ID NO:36 and (b) comprises the substitution F48A/Lrelative to SEQ ID NO:36. Some such polypeptides comprise a sequencewhich differs from SEQ ID NO:36 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or 16 amino acid positions, e.g., in 1-15 amino acidpositions, in 1-14 amino acid positions, in 1-13 amino acid positions,in 1-12 amino acid positions, 1-11 amino acid positions, in 1-10 aminoacid positions, in 1-9 amino acid positions, in 1-8 amino acidpositions, in 1-7 amino acid positions, in 1-6 amino acid positions, in1-5 amino acid positions, in 1-4 amino acid positions, in 1-3 amino acidpositions, or in 1-2 amino acid positions. Some such polypeptidescomprise the substitution F48A relative to SEQ ID NO:36. In someinstances, the polypeptide further comprises one or more substitution(s)relative to SEQ ID NO:36 selected from the group consisting of M21A;I24P; P26L; H47Q; T51V/P; S55P/F/A; V56L; H58Y; L60M; F65A; F68P;F90Y/A; M93L/P; N95D; L111A; N113K; V114E/P; F124A; R125Q; I127P; T132K;L154F; and E160D. In some instances, the polypeptide exhibits aninterferon-alpha activity (e.g., antiviral activity, T_(H)1differentiation activity, and/or antiproliferative activity). Some suchpolypeptides further comprise one or more additional amino acid(s), suchas a methionine added to the N-terminus. The invention also providesfusion proteins and conjugates comprising these polypeptides, andisolated or recombinant nucleic acids encoding these polypeptides.

The invention also provides isolated or recombinant polypeptides whicheach comprise a sequence which (a) differs in 1 to 16 amino acidpositions from SEQ ID NO:36 and (b) comprises the substitution F90Arelative to SEQ ID NO:36. Some such polypeptides comprise a sequencewhich differs from SEQ ID NO:36 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or 16 amino acid positions, e.g., in 1-15 amino acidpositions, in 1-14 amino acid positions, in 1-13 amino acid positions,in 1-12 amino acid positions, 1-11 amino acid positions, in 1-10 aminoacid positions, in 1-9 amino acid positions, in 1-8 amino acidpositions, in 1-7 amino acid positions, in 1-6 amino acid positions, in1-5 amino acid positions, in 1-4 amino acid positions, in 1-3 amino acidpositions, or in 1-2 amino acid positions. In some instances, thepolypeptide further comprises one or more substitution(s) relative toSEQ ID NO:36 selected from the group consisting of M21A; I24P; P26L;H47Q; F48A/L; T51V/P; S55P/F/A; V56L; H58Y; L60M; F65A; F68P; M93L/P;N95D; L111A; N113K; V114E/P; F124A; R125Q; I127P; T132K; L154F; andE160D. In some instances, the polypeptide exhibits an interferon-alphaactivity (e.g., antiviral activity, T_(H)1 differentiation activity,and/or antiproliferative activity). Some such polypeptides furthercomprise one or more additional amino acid(s), such as a methionineadded to the N-terminus. The invention also provides fusion proteins andconjugates comprising these polypeptides, and isolated or recombinantnucleic acids encoding these polypeptides.

The invention also provides isolated or recombinant polypeptides whicheach comprise a sequence which (a) differs in 1 to 16 amino acidpositions from SEQ ID NO:36 and (b) comprises the substitution E160Drelative to SEQ ID NO:36. Some such polypeptides comprise a sequencewhich differs from SEQ ID NO:36 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or 16 amino acid positions, e.g., in 1-15 amino acidpositions, in 1-14 amino acid positions, in 1-13 amino acid positions,in 1-12 amino acid positions, 1-11 amino acid positions, in 1-10 aminoacid positions, in 1-9 amino acid positions, in 1-8 amino acidpositions, in 1-7 amino acid positions, in 1-6 amino acid positions, in1-5 amino acid positions, in 1-4 amino acid positions, in 1-3 amino acidpositions, or in 1-2 amino acid positions. In some instances, thepolypeptide further comprises one or more substitution(s) relative toSEQ ID NO:36 selected from the group consisting of M21A; I24P; P26L;H47Q; F48A/L; T51V/P; S55P/F/A; V56L; H58Y; L60M; F65A; F68P; F90Y/A;M93L/P; N95D; L111A; N113K; V114E/P; F124A; R125Q; I127P; T132K; andL154F. In some instances, the polypeptide exhibits an interferon-alphaactivity (e.g., antiviral activity, T_(H)1 differentiation activity,and/or antiproliferative activity). Some such polypeptides furthercomprise one or more additional amino acid(s), such as a methionineadded to the N-terminus. The invention also provides fusion proteins andconjugates comprising these polypeptides, and isolated or recombinantnucleic acids encoding these polypeptides.

The invention also provides isolated or recombinant polypeptides whicheach comprise a sequence which (a) differs in 0 to 16 amino acidpositions from SEQ ID NO:38 and (b) comprises one or more of: Ala atposition 21; Pro at position 24; Ala or Leu at position 48; Pro atposition 51; Ala at position 55; Ala at position 65; Pro at position 68;Ala at position 90; Pro at position 93; Ala at position 111; Pro atposition 114; Ala at position 124; Pro at position 127; and Glu atposition 160 (position numbering relative to SEQ ID NO:38). Some suchpolypeptides comprise a sequence which differs from SEQ ID NO:38 in 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acidpositions, e.g., in 0-15 amino acid positions, in 0-14 amino acidpositions, in 0-13 amino acid positions, in 0-12 amino acid positions,in 0-11 amino acid positions, in 0-10 amino acid positions, in 0-9 aminoacid positions, in 0-8 amino acid positions, in 0-7 amino acidpositions, in 0-6 amino acid positions, in 0-5 amino acid positions, in0-4 amino acid positions, in 0-3 amino acid positions, in 0-2 amino acidpositions, or in 0-1 amino acid positions. Some such polypeptidescomprise an Ala or a Leu at position 48. Some such polypeptides comprisean Ala at position 48. In some instances, the polypeptide furthercomprises one or more substitution(s) relative to SEQ ID NO:38 selectedfrom the group consisting of P26L; H47Q; V51T; F55P/S; L56V; Y58H; L60M;F90Y; M93L; N95D; N113K; V114E; R125Q; T132K; and F154L. In someinstances, the polypeptide exhibits an interferon-alpha activity (e.g.,antiviral activity, T_(H)1 differentiation activity, and/orantiproliferative activity). Some such polypeptides further comprise oneor more additional amino acid(s), such as a methionine added to theN-terminus. The invention also provides fusion proteins and conjugatescomprising these polypeptides, and isolated or recombinant nucleic acidsencoding these polypeptides.

Other modifications contemplated for polypeptides of the inventioninclude those described below and in the section entitled“INTERFERON-ALPHA CONJUGATES”.

It is to be understood that while the examples and modifications to theparent polypeptide are generally provided herein relative to thesequence SEQ ID NO:1 (or relative to SEQ ID NO:36, or relative to someother specified sequence), the disclosed modifications may also be madein equivalent amino acid positions of any of the other polypeptides ofthe invention (such as a polypeptide comprising a sequence selected fromSEQ ID NOs:2-35 and SEQ ID NOs:37-44) described herein. Thus, as anexample, the substitution F48L relative to SEQ ID NO:1 is understood tocorrespond to the substitution A48L in SEQ ID NO:13, and so on.

The following tables provide sequences of some interferon-alphapolypeptides of the invention. For clarity, the sequences are shownrelative to reference sequences SEQ ID NO:1 (Table 1) or SEQ ID NO:36(Table 2). Some such polypeptides exhibit an interferon-alpha activity,such as antiviral activity, T_(H)1 differentiation activity, and/orantiproliferative activity. TABLE 1 Polypeptide Sequence (relative toSEQ ID NO: 1) Clone name SEQ ID SEQ ID NO: 1 B9x14EC4 SEQ ID NO: 1 SEQID NO: 1 F55P 14epi07 SEQ ID NO: 2 SEQ ID NO: 1 14epi08 SEQ ID NO: 3V51A Q52P F55S L56V F57L Y58H SEQ ID NO: 1 V51A Q52E A53T F55P L56V F57LY58H 14epi09 SEQ ID NO: 4 SEQ ID NO: 1 V51A Q52E F55S L56V F57L Y58H14epi10 SEQ ID NO: 5 SEQ ID NO: 1 14epi11 SEQ ID NO: 6 V51A F55S L56AY58H SEQ ID NO: 1 F48L E160D 14epi12d SEQ ID NO: 7 SEQ ID NO: 1 14epi13SEQ ID NO: 8 H47Q SEQ ID NO: 1 M61I 14epi14 SEQ ID NO: 9 SEQ ID NO: 1F48L 14epi15 SEQ ID NO: 10 SEQ ID NO: 1 F48L F55A 14epi16 SEQ ID NO: 11SEQ ID NO: 1 F48L F55S 14epi17 SEQ ID NO: 12 SEQ ID NO: 1 F48A 14epi18SEQ ID NO: 13 SEQ ID NO: 1 F48A F55A 14epi19 SEQ ID NO: 14 SEQ ID NO: 1F48A F55S 14epi20 SEQ ID NO: 15 SEQ ID NO: 1 F55A 14epi21 SEQ ID NO: 16SEQ ID NO: 1 F55S 14epi22 SEQ ID NO: 17 SEQ ID NO: 1 F48A F55S L111A14epi23 SEQ ID NO: 18 SEQ ID NO: 1 F48A F55S N113K V114E 14epi24 SEQ IDNO: 19 SEQ ID NO: 1 F48L F55S L111A 14epi25 SEQ ID NO: 20 SEQ ID NO: 1F48L F55S N113K V114E 14epi26 SEQ ID NO: 21 SEQ ID NO: 1 F48A F55S F65AL111A 14epi27 SEQ ID NO: 22 SEQ ID NO: 1 F48A F55S F65A N113K V114E14epi28 SEQ ID NO: 23 SEQ ID NO: 1 F48A F55S F65A L111A 14epi29 SEQ IDNO: 24 SEQ ID NO: 1 F48A F55A L111A 14epi30 SEQ ID NO: 25 SEQ ID NO: 1F48A F55A N113K V114E 14epi31 SEQ ID NO: 26 SEQ ID NO: 1 F48L Q52P F55SN113K V114E 14epi32 SEQ ID NO: 27 SEQ ID NO: 1 Q52P F55S N113K V114E14epi33 SEQ ID NO: 28 SEQ ID NO: 1 Q52P F55S N113K V114E 14epi34 SEQ IDNO: 29 SEQ ID NO: 1 F48L V51A Q52P F55S L56V F57L Y58H 14epi35 SEQ IDNO: 30 N113K V114E SEQ ID NO: 1 V51A Q52E A53T F55P L56V F57L Y58H14epi36 SEQ ID NO: 31 N113K V114E SEQ ID NO: 1 V51A F55S L56V F57L Y58HN113K 14epi37 SEQ ID NO: 32 V114E SEQ ID NO: 1 V51A Q52P F55S L56V F57LY58H 14epi38 SEQ ID NO: 33 N113K V114E SEQ ID NO: 1 H47Q F48A F55S14epi39 SEQ ID NO: 34 SEQ ID NO: 1 H47Q F48A F55S L56V Y58H 14epi42 SEQID NO: 35

TABLE 2 Polypeptide Sequence (relative to SEQ ID NO: 36) Clone name SEQID SEQ ID NO: 36 25epi08 SEQ ID NO: 36 SEQ ID NO: 36 F48A L154F 25epi18SEQ ID NO: 37 SEQ ID NO: 36 F48A T51V S55F 25epi19 SEQ ID NO: 38 V56LH58Y L154F SEQ ID NO: 36 F48A T51V V56L 25epi20 SEQ ID NO: 39 H58Y L154FSEQ ID NO: 36 F48A T51V L154F 25epi25 SEQ ID NO: 40 SEQ ID NO: 36 F48AT51V F90Y 25epi26 SEQ ID NO: 41 M93L N95D L154F SEQ ID NO: 36 F48A T51VF90A 25epi27 SEQ ID NO: 42 R125Q L154F SEQ ID NO: 36 F48A T51V F90Y25epi28 SEQ ID NO: 43 M93L N95D R125Q L154F SEQ ID NO: 36 L154F E160D25epi29 SEQ ID NO: 44Sequence Variations

As noted above, polypeptides of the present invention includepolypeptides comprising sequences which differ from SEQ ID NO:1 in 1 to16 amino acid positions and comprise one or more substitutions relativeto SEQ ID NO:1 selected from the group consisting of F48A/L; V51P; F55A;F65A; F68P; L111A; and V114P. Polypeptides of the present invention alsoinclude polypeptides comprising sequences which differ from SEQ ID NO:36in 1 to 16 amino acid positions and comprise one or more substitutionsrelative to SEQ ID NO:36 selected from the group consisting of M21A;I24P; F48A/L; T51P; S55A; F65A; F68P; F90A; M93P; L111A; V114P; F124A;I127P; and E160D. Some such polypeptides exhibit an interferon-alphaactivity.

For example, some polypeptides of the invention comprise a sequencehaving a length of about 151 amino acids, such as about 151, 152, 153,154, 155 ,156, 157, 158, 159, 160, 161, 162, 163, 164, or 165 aminoacids, corresponding to a deletion of between 1 and 15 amino acidsrelative to a parent polypeptide sequence (such as, for example, one ofSEQ ID NOs:2-35 or 37-44). In some instances, between 1 and 11, e.g.,between 1 and 10, such as between 1 and 7, e.g. between 1 and 5, such asbetween 1 and 3 amino acids are deleted from the C-terminus, i.e. thepolypeptide is C-terminally truncated compared to the parent polypeptidesequence (such as, for example, one of SEQ ID NOs: 2-35 or 37-44) by1-11 amino acid residues (e.g. by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11amino acid residues), such as by 1-10, 1-7, e.g., by 1-5 or by 1-3 aminoacid residues. Alternatively, or in addition, some such polypeptides areN-terminally truncated compared to the parent polypeptide sequence (suchas, one of SEQ ID NOs: 2-35 or 37-44) by 1-4 amino acid residues (e.g.by 1, 2, 3, or 4 amino acid residues), e.g., 1-4, 1-3, 1-2 or 1 aminoacid residue(s) are removed from the N-terminus. Some such polypeptidesfurther comprise a methionine at the N-terminus. Some such polypeptidesexhibit an interferon-alpha activity.

As another example, some polypeptides of the invention comprise asequence containing between 1 and 16 amino acid substitutions relativeto SEQ ID NO:1 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,or 16 amino acid substitutions), such as 1-14 or 1-12 or 1-10 or 1-8 or1-6 or 1-5 or 1-4 or 1-3 or 1-2 amino acid substitutions, in which atleast one of the substitutions is selected from the group consisting ofF48A/L; V51P; F55A; F65A; F68P; L111A; and V114P. Other polypeptides ofthe invention comprise a sequence containing between 1 and 16 amino acidsubstitutions relative to SEQ ID NO:36 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, or 16 amino acid substitutions), such as 1-14 or1-12 or 1-10 or 1-8 or 1-6 or 1-5 or 1-4 or 1-3 or 1-2 amino acidsubstitutions, in which at least one of the substitutions is selectedfrom the group consisting of M21A; I24P; F48A/L; T51P; S55A; F65A; F68P;F90A; M93P; L111A; V114P; F124A; I127P; and E160D. One or moreadditional amino acid substitutions may be made in the polypeptidesequence according to, for example, a substitution group (such as, aconservative substitution group), such as one set forth below.Alternatively, or in addition, one or more additional amino acidsubstitutions may made in the polypeptide sequence which introduces orremoves an amino acid residue comprising an attachment group for anon-polypeptide moiety. Examples include introduction of one or moreN-glycosylation site(s), introduction of one or more cysteine residue(s)or lysine residue(s), removal of one or more N-glycosylation site(s),and/or or removal of one or more lysine(s) or histidine(s). Some suchpolypeptides exhibit an interferon-alpha activity.

As a non-limiting example, a polypeptide of the invention may have asequence which differs from SEQ ID NO:1 in a total of up to 16 positions(which may be a combination of amino acid substitutions, deletions,and/or insertions, including those described above). In some instances,none, some, or all of the substitutions are substitutions according to asubstitution group defined below.

Amino acid substitutions in accordance with the invention may include,but are not limited to, one or more conservative amino acidsubstitutions. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. One example is providedin the table below (Table 3), which sets forth six exemplary groups thatcontain amino acids which may be considered “conservative substitutions”for one another. TABLE 3 Conservative Substitution Groups 1 Alanine (A)Glycine (G) Serine (S) Threonine (T) 2 Aspartic acid (D) Glutamic acid(E) 3 Asparagine (N) Glutamine (Q) 4 Arginine (R) Lysine (K) Histidine(H) 5 Isoleucine (I) Leucine (L) Methionine (M) Valine (V) 6Phenylalanine (F) Tyrosine (Y) Tryptophan (W)

Other substitution groups of amino acids can be envisioned. For example,amino acids can be grouped by similar function or chemical structure orcomposition (e.g., acidic, basic, aliphatic, aromatic,sulfur-containing). For example, an Aliphatic grouping may comprise:Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I). Othergroups containing amino acids that are considered conservativesubstitutions for one another include: Aromatic: Phenylalanine (F),Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M),Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine (H); Acidic:Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q). Seealso Creighton (1984) Proteins, W.H. Freeman and Company, for additionalgroupings of amino acids. Listing of a polypeptide sequence herein, inconjunction with the above substitution groups, provides an expresslisting of all conservatively substituted polypeptide sequences.

Percent Sequence Identity

In one aspect, the invention provides isolated or recombinantpolypeptides each comprising a sequence having at least 90% sequenceidentity (e.g., at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99% amino acidsequence identity) to any one of SEQ ID NOs:1-35, wherein the sequencecomprises one or more of: Ala or Leu at position 48; Pro at position 51;Ala at position 55; Ala at position 65; Pro at position 68; Ala atposition 111; and Pro at position 114, amino acid position relative toSEQ ID NO:1. In some instances the polypeptide exhibits aninterferon-alpha activity.

The invention also provides isolated or recombinant polypeptides eachcomprising a sequence having at least 90% sequence identity (e.g., atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, or at least about 99% amino acid sequence identity) toany one of SEQ ID NOs:36-44, wherein the sequence comprises one or moreof: Ala at position 21; Pro at position 24; Ala or Leu at position 48;Pro at position 51; Ala at position 55; Ala at position 65; Pro atposition 68; Ala at position 90; Pro at position 93; Ala at position111; Pro at position 114; Ala at position 124; Pro at position 127; andAsp at position 160, amino acid position relative to SEQ ID NO:36. Insome instances the polypeptide exhibits an interferon-alpha activity.

The degree to which a sequence (polypeptide or nucleic acid) is similarto another provides an indication of similar structural and functionalproperties for the two sequences. Accordingly, in the context of thepresent invention, sequences which have a similar sequence to any givenexemplar sequence are a feature of the present invention. In particular,sequences that have percent sequence identities as defined below are afeature of the invention.

A variety of methods of determining sequence relationships can be used,including manual alignment and computer assisted sequence alignment andanalysis. A variety of computer programs for performing sequencealignments are available, or an alignment can be prepared manually byone of skill, as described below.

As noted above, the sequences of the polypeptides and nucleic acidsemployed in the subject invention need not be identical, but can besubstantially identical to the corresponding sequence of a polypeptideof the invention or nucleic acid of the invention. For example,polypeptides of the invention can be subject to various changes, such asone or more amino acid insertions, deletions, and/or substitutions,either conservative or non-conservative, including where, e.g., suchchanges might provide for certain advantages in their use, such as, intheir therapeutic or prophylactic use or administration or diagnosticapplication. The nucleic acids of the invention can also be subject tovarious changes, such as one or more substitutions of one or morenucleic acids in one or more codons such that a particular codon encodesthe same or a different amino acid, resulting in either a silentvariation (as defined herein) or non-silent variation, or one or moredeletions of one or more nucleic acids (or codons) in the sequence. Thenucleic acids can also be modified to include one or more codons thatprovide for optimum expression in an expression system (e.g., bacterialor mammalian), while, if desired, said one or more codons still encodethe same amino acid(s). Such nucleic acid changes might provide forcertain advantages in their therapeutic or prophylactic use oradministration, or diagnostic application. The nucleic acids andpolypeptides can be modified in a number of ways so long as theycomprise a sequence substantially identical (as defined below) to asequence in a respective nucleic acid or polypeptide of the invention.

The term “identical” or “identity,” in the context of two or morenucleic acid or polypeptide sequences, refers to two or more sequencesthat are the same or have a specified percentage of amino acid residuesor nucleotides that are the same, when compared and aligned for maximumsimilarity, as determined using the sequence comparison algorithmdescribed below or by visual inspection.

The “percent sequence identity” (“% identity”) of a subject sequence toa reference (i.e. query) sequence means that the subject sequence isidentical (i.e., on an amino acid-by-amino acid basis for a polypeptidesequence, or a nucleotide-by-nucleotide basis for a polynucleotidesequence) by a specified percentage to the query sequence over acomparison length.

The percent sequence identity of a subject sequence to a query sequenceis calculated as follows. First, the optimal alignment of the twosequences is determined using a sequence comparison algorithm withspecific alignment parameters. This determination of the optimalalignment may be performed using a computer, or may be manuallycalculated, as described below. Then, the two optimally alignedsequences are compared over the comparison length, and the number ofpositions in the optimal alignment at which identical residues occur inboth sequences are determined, which provides the number of matchedpositions. The number of matched positions is then divided by the totalnumber of positions of the comparison length (which, unless otherwisespecified, is the length of the query sequence), and then the result ismultiplied by 100, to yield the percent sequence identity of the subjectsequence to the query sequence.

With regard to polypeptide sequences, typically one sequence is regardedas a “query sequence” (for example, a polypeptide sequence of theinvention) to which one or more other sequences, i.e., “subjectsequence(s)” (for example, sequences present in a sequence database) arecompared. The sequence comparison algorithm uses the designatedalignment parameters to determine the optimal alignment between thequery sequence and the subject sequence(s). When comparing a querysequence against a sequence database, such as, e.g., GENBANK® (GeneticSequence Data Bank; U.S. Department of Health and Human Services) orGENESEQ® (Thomson Derwent; also available as DGENE® on STN), usuallyonly the query sequence and the alignment parameters are input into thecomputer; optimal alignments between the input query sequence and eachsubject sequence present in the database are returned, generally for upto a desired number of subject sequences.

Two polypeptide sequences are “optimally aligned” when they are alignedusing defined parameters, i.e., a defined amino acid substitutionmatrix, gap existence penalty (also termed gap open penalty), and gapextension penalty, so as to arrive at the highest similarity scorepossible for that pair of sequences. The BLOSUM62 matrix (Henikoff andHenikoff (1992) Proc. Natl. Acad. Sci. USA 89(22):10915-10919) is oftenused as a default scoring substitution matrix in polypeptide sequencealignment algorithms (such as BLASTP, described below). The gapexistence penalty is imposed for the introduction of a single amino acidgap in one of the aligned sequences, and the gap extension penalty isimposed for each residue position in the gap. Unless otherwise stated,alignment parameters employed herein are: BLOSUM62 scoring matrix, gapexistence penalty=11, and gap extension penalty=1. The alignment scoreis defined by the amino acid positions of each sequence at which thealignment begins and ends (e.g. the alignment window), and optionally bythe insertion of a gap or multiple gaps into one or both sequences, soas to arrive at the highest possible similarity score.

While optimal alignment between two or more sequences can be determinedmanually (as described below), the process is facilitated by the use ofa computer-implemented alignment algorithm such as BLAST® (NationalLibrary of Medicine), e.g., BLASTP for polypeptide sequences and BLASTNfor nucleic acid sequences, described in Altschul et al. (1997) NucleicAcids Res. 25:3389-3402, and made available to the public throughvarious sources, such as the National Center for BiotechnologyInformation (NCBI) Website. When using a computerized BLAST interface,if the option exists to use a “low complexity filter”, this optionshould be turned off (i.e., no filter).

The optimal alignment between two polypeptide sequences can also bedetermined by a manual calculation of the BLASTP algorithm (i.e.,without aid of a computer) using the same alignment parameters specifiedabove (matrix=BLOSUM62, gap open penalty=11, and gap extensionpenalty=1). To begin, the two sequences are initially aligned by visualinspection. An initial alignment score is then calculated as follows:for each individual position of the alignment (i.e., for each pair ofaligned residues), a numerical value is assigned according to theBLOSUM62 matrix (FIG. 4). The sum of the values assigned to each pair ofresidues in the alignment is the initial alignment score. If the twosequences being aligned are highly similar, often this initial alignmentprovides the highest possible alignment score. The alignment with thehighest possible alignment score is the optimal alignment based on thealignment parameters employed. FIG. 5A shows an example of thecalculation of an alignment score for two sequences, a “query” sequence,identified herein as residues 29-50 of SEQ ID NO:1 (upper), and a“subject” sequence, identified herein as residues 30-52 of SEQ ID NO:8(lower). The sequences were aligned by visual inspection, and thenumerical value assigned by the BLOSUM62 matrix for each aligned pair ofamino acids is shown beneath each position in the alignment (to aid invisualization, each identical pair of amino acids in the alignment isshown in boldface). In this example, this initial alignment provided thehighest possible alignment score (the sum of the values shown beneatheach aligned position); any other alignment of these two sequences, withor without gaps, would result in a lower alignment score.

In some instances, a higher alignment score might be obtained byintroducing one or more gaps into the alignment. Whenever a gap isintroduced into an alignment, a gap open penalty is assigned, and inaddition a gap extension penalty is assessed for each residue positionwithin that gap. Therefore, using the alignment parameters describedabove (including gap open penalty=11 and gap extension penalty=1), a gapof one residue in the alignment would correspond to a value of−(11+(1×1))=−12 assigned to the gap; a gap of three residues wouldcorrespond to a value of −(11+(3×1))=−14 assigned to the gap, and so on.This calculation is repeated for each new gap introduced into thealignment. FIGS. 5B and 5C show an example which demonstrates howintroduction of a gap into an alignment can result in a higher alignmentscore, despite the gap penalty. FIG. 5B shows an initial alignment ofresidues 29-50 of SEQ ID NO:1 (upper, query) and residues 30-50 of SEQID NO:46 (lower, subject) made by visual inspection, which results in aninitial alignment score of 67. FIG. 5C shows the effect of theintroduction of a one-residue gap in SEQ ID NO:46 on the alignmentscore; despite the gap penalty of −12, the overall alignment score ofthe two sequences increases to 88. In this example, the alignment shownin FIG. 5C provides the highest possible alignment score, and is thusthe optimal alignment of these two sequences; any other alignment ofthese two sequences (with or without gaps) would result in a loweralignment score.

It is to be understood that the examples of sequence alignmentcalculations described above, which use relatively short sequences, areprovided for illustrative purposes only; in practice, the alignmentparameters employed (BLOSUM62 matrix, gap open penalty=11, and gapextension penalty=1) are generally intended for polypeptide sequences 85amino acids in length or longer. The NCBI website provides the followingalignment parameters for sequences of other lengths (which are suitablefor computer-aided as well as manual alignment calculation, using thesame procedure as described above). For sequences of 50-85 amino acidsin length, optimal parameters are BLOSUM80 matrix (Henikoff andHenikoff, supra), gap open penalty=10, and gap extension penalty=1. Forsequences of 35-50 amino acids in length, optimal parameters are PAM70matrix (Dayhoff, M. O., Schwartz, R. M. & Orcutt, B. C. (1978) “A modelof evolutionary change in proteins.” In Atlas of Protein Sequence andStructure, vol. 5, suppl. 3, M. O. Dayhoff (ed.), pp. 345-352, Natl.Biomed. Res. Found., Washington, D.C.), gap open penalty=10, and gapextension penalty=1. For sequences of less than 35 amino acids inlength, optimal parameters are PAM30 matrix (Dayhoff, M. O., supra), gapopen penalty=9, and gap extension penalty=1.

Once the sequences are optimally aligned, the percent identity of thesubject sequence relative to the query sequence is calculated bycounting the number of positions in the optimal alignment which containidentical residue pairs, divide that by the number of residues in thecomparison length (which, unless otherwise specified, is the number ofresidues in the query sequence), and multiplying the resulting number by100. Referring back to the examples shown in FIG. 5, in each example thesequence designated as the query sequence is 22 amino acids in length.In the alignment of FIG. 5A, 20 pairs of aligned amino acid residues(shown in boldface) are identical in the optimal alignment of the querysequence (upper) with the subject sequence (lower). Thus, thisparticular subject sequence has (20/22)×100=91.1% identity to the querysequence; in other words, the subject sequence in the alignment of FIG.5A has at least 91% amino acid sequence identity to the query sequence.In the alignment shown in FIG. 5C, 18 pairs of amino acid residues(shown in boldface) in the optimal alignment are identical; thus thisparticular subject sequence has (18/22)×100=81.8% identity to the querysequence; in other words, the subject sequence in the alignment of FIG.5C has at least 81% amino acid sequence identity to the query sequence.

As applied to polypeptides, the term “substantial identity” (or“substantially identical”) typically means that when two amino acidsequences (i.e. a query sequence and a subject sequence) are optimallyaligned using the BLASTP algorithm (manually or via computer) usingappropriate parameters described above, the subject sequence has atleast about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more percent amino acid sequence identity to the querysequence. In some instances, the substantial identity exists over acomparison length of at least about 100 amino acid residues, such as, atleast about 110, 120, 125, 130, 135, 140, 145, 150, 155, 156, 157, 158,159, 160, 161, 162, 163, 164, 165, or 166 amino acid residues.Similarly, as applied in the context of two nucleic acid sequences, theterm substantial identity (or substantially identical) means that whentwo nucleic acid sequences (i.e. a query and a subject sequence) areoptimally aligned using the BLASTN algorithm (manually or via computer)using appropriate parameters described below, the subject sequence hasat least about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more percent nucleic acid sequence identity tothe query sequence. Parameters used for nucleic acid sequence alignmentsare: match reward 1, mismatch penalty −3, gap existence penalty 5, gapextension penalty 2 (substitution matrices are not used in the BLASTNalgorithm). In some instances, the substantial identity exists over acomparison length of at least about 300 nucleotide residues, such as atleast about 330, 360, 375, 390, 405, 420, 435, 450, 465, 480, 483, 486,489, 492, 495, or 498 nucleotides.

Additional Aspects

Any polypeptide of the invention may be present as part of a largerpolypeptide sequence, e.g. a fusion protein, such as occurs upon theaddition of one or more domains or subsequences for stabilization ordetection or purification of the polypeptide. A polypeptide purificationsubsequence may include, e.g., an epitope tag, a FLAG tag, apolyhistidine sequence, a GST fusion, or any otherdetection/purification subsequence or “tag” known in the art. Theseadditional domains or subsequences either have little or no effect onthe activity of the polypeptide of the invention, or can be removed bypost synthesis processing steps such as by treatment with a protease,inclusion of an intein, or the like.

The invention includes fusion proteins comprising a polypeptide of theinvention, e.g., as described herein, fused to an Ig molecule, e.g., ahuman IgG Fc (“fragment crystallizable,” or fragment complement binding)hinge, CH2 domain and CH3 domain, and nucleotide sequences encoding suchfusion protein. Fc is the portion of the antibody responsible forbinding to antibody receptors on cells and the C1q component ofcomplement. These fusion proteins and their encoding nucleic acids areuseful as prophylactic and/or therapeutic drugs or as diagnostic tools(see also, e.g., Challita-Eid, P. et al. (1998) J. Immunol160:3419-3426; Sturmhoefel, K. et al. (1999) Cancer Res 59:4964-4972).The invention also includes fusion proteins comprising a polypeptide ofthe invention, fused to an albumin molecule, such as human serum albumin(HSA), as described, for example, in U.S. Pat. No. 5,876,969, andnucleotide sequences encoding the fusion protein. The Ig and albuminfusion proteins may exhibit increased polypeptide serum half-life and/orfunctional in vivo half-life, reduced polypeptide antigenicity,increased polypeptide storage stability, or increasing bioavailability,e.g. increased AUC_(sc), and are thus may be useful as prophylacticand/or therapeutic drugs.

Any polypeptide of the invention may also comprise one or more modifiedamino acid. The modified amino acid may be, e.g., a glycosylated aminoacid, a PEGylated amino acid, a farnesylated amino acid, an acetylatedamino acid, a biotinylated amino acid, an amino acid conjugated to alipid moiety, or an amino acid conjugated to an organic derivatizingagent. The presence of modified amino acids may be advantageous in, forexample, (a) increasing polypeptide serum half-life and/or functional invivo half-life, (b) reducing polypeptide antigenicity, (c) increasingpolypeptide storage stability, or (d) increasing bioavailability, e.g.increasing the AUC_(sc). Amino acid(s) are modified, for example,co-translationally or post-translationally during recombinant production(e.g., N-linked glycosylation at N-X-S/T motifs during expression inmammalian cells) or modified by synthetic means. This aspect isdescribed in more detail in the section herein entitled“INTERFERON-ALPHA CONJUGATES”.

The invention also provides a composition comprising at least onepolypeptide of the invention, and an excipient or carrier. Thecomposition may be a composition comprising a pharmaceuticallyacceptable excipient or carrier. Exemplary compositions and excipientsand carriers are described below.

Making Polypeptides

Recombinant methods for producing and isolating polypeptides of theinvention are described herein. In addition to recombinant production,the polypeptides may be produced by direct peptide synthesis usingsolid-phase techniques (see, e.g., Stewart et al. (1969) Solid-PhasePeptide Synthesis, WH Freeman Co, San Francisco; Merrifield J. (1963) JAm Chem Soc 85:2149-2154). Peptide synthesis may be performed usingmanual techniques or by automation. Automated synthesis may be achieved,for example, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer, Foster City, Calif.) in accordance with the instructions providedby the manufacturer. For example, subsequences may be chemicallysynthesized separately and combined using chemical methods to providefull-length polypeptides or fragments thereof. Alternatively, suchsequences may be ordered from any number of companies which specializein production of polypeptides. Most commonly, polypeptides of theinvention may be produced by expressing coding nucleic acids andrecovering polypeptides, e.g., as described below.

Methods for producing the polypeptides of the invention are alsoincluded. One such method comprises introducing into a population ofcells any nucleic acid of the invention described herein, which isoperatively linked to a regulatory sequence effective to produce theencoded polypeptide, culturing the cells in a culture medium to expressthe polypeptide, and isolating the polypeptide from the cells or fromthe culture medium. An amount of nucleic acid sufficient to facilitateuptake by the cells (transfection) and/or expression of the polypeptideis utilized. The nucleic acid is introduced into such cells by anydelivery method described herein, including, e.g., injection, gene gun,passive uptake, etc. The nucleic acid may be part of a vector, such as arecombinant expression vector, including a DNA plasmid vector, or anyvector described herein. The nucleic acid or vector comprising a nucleicacid of the invention may be prepared and formulated as describedherein, above. Such a nucleic acid or expression vector may beintroduced into a population of cells of a mammal in vivo, or selectedcells of the mammal (e.g., tumor cells) may be removed from the mammaland the nucleic acid expression vector introduced ex vivo into thepopulation of such cells in an amount sufficient such that uptake andexpression of the encoded polypeptide results. Or, a nucleic acid orvector comprising a nucleic acid of the invention is produced usingcultured cells in vitro. In one aspect, the method of producing apolypeptide of the invention comprises introducing into a population ofcells a recombinant expression vector comprising any nucleic acid of theinvention described herein in an amount and formula such that uptake ofthe vector and expression of the encoded polypeptide will result;administering the expression vector into a mammal by anyintroduction/delivery format described herein; and isolating thepolypeptide from the mammal or from a byproduct of the mammal.

Antibodies

In another aspect of the invention, a polypeptide of the invention (oran antigenic fragment thereof) is used to produce antibodies which have,e.g., diagnostic, therapeutic, or prophylactic uses, e.g., related tothe activity, distribution, and expression of polypeptides and fragmentsthereof. Antibodies to polypeptides of the invention may be generated bymethods well known in the art. Such antibodies may include, but are notlimited to, polyclonal, monoclonal, chimeric, humanized, single chain,Fab fragments and fragments produced by a Fab expression library.Antibodies, e.g., those that block receptor binding, are especiallypreferred for therapeutic and/or prophylactic use.

Polypeptides for antibody induction do not require biological activity;however, the polypeptides or peptides should be antigenic. Peptides usedto induce specific antibodies may have an amino acid sequence consistingof at least about 10 amino acids, preferably at least about 15 or 20amino acids or at least about 25 or 30 amino acids. Short stretches of apolypeptide may be fused with another protein, such as keyhole limpethemocyanin, and antibody produced against the chimeric molecule.

Methods of producing polyclonal and monoclonal antibodies are known tothose of skill in the art, and many antibodies are available. See, e.g.,Current Protocols in Immunology, John Colligan et al., eds., Vols. I-IV(John Wiley & Sons, Inc., NY, 1991 and 2001 Supplement); and Harlow andLane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press,NY; Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) LangeMedical Publications, Los Altos, Calif., and references cited therein;and Goding (1986) Monoclonal Antibodies: Principles and Practice (2ded.) Academic Press, New York, N.Y.; and Kohler and Milstein (1975)Nature 256:495-497. Other suitable techniques for antibody preparationinclude selection of libraries of recombinant antibodies in phage orsimilar vectors. See, Huse et al. (1989) Science 246:1275-1281; and Wardet al. (1989) Nature 341:544-546. Specific monoclonal and polyclonalantibodies and antisera will usually bind with a K_(D) of at least about0.1 μM, preferably at least about 0.01 μM or better, and most typicallyand preferably, 0.001 μM or better.

Detailed methods for preparation of chimeric (humanized) antibodies canbe found in U.S. Pat. No. 5,482,856. Additional details on humanizationand other antibody production and engineering techniques can be found inBorrebaeck (ed.) (1995) Antibody Engineering, 2^(nd) Edition Freeman andCompany, NY (Borrebaeck); McCafferty et al. (1996) Antibody Engineering,A Practical Approach IRL at Oxford Press, Oxford, England (McCafferty),and Paul (1995) Antibody Engineering Protocols Humana Press, Towata,N.J. (Paul).

In one aspect, this invention provides for fully humanized antibodiesagainst the polypeptides of the invention or fragments thereof.Humanized antibodies are especially desirable in applications where theantibodies are used as therapeutics and/or prophylactics in vivo inhuman patients. Human antibodies consist of characteristically humanimmunoglobulin sequences. The human antibodies of this invention can beproduced in using a wide variety of methods (see, e.g., Larrick et al.,U.S. Pat. No. 5,001,065, and Borrebaeck McCafferty and Paul, supra, fora review). In one aspect, the human antibodies of the present inventionare produced initially in trioma cells. Genes encoding the antibodiesare then cloned and expressed in other cells, such as nonhuman mammaliancells. The general approach for producing human antibodies by triomatechnology is described by Ostberg et al. (1983), Hybridoma 2:361-367,Ostberg, U.S. Pat. No. 4,634,664, and Engelman et al., U.S. Pat. No.4,634,666. The antibody-producing cell lines obtained by this method arecalled triomas because they are descended from three cells—two human andone mouse. Triomas have been found to produce antibody more stably thanordinary hybridomas made from human cells.

Other uses contemplated for polypeptides of the invention are providedthroughout the specification.

Interferon-Alpha Conjugates

In another aspect, the invention relates to a conjugate comprising apolypeptide exhibiting an interferon-alpha activity which comprises anamino acid sequence of any one of SEQ ID NOs:2-35 and SEQ ID NOs:37-44,and at least one non-polypeptide moiety attached to the polypeptide,such as e.g., 1-6, 1-5, 1-4, 1-3, e.g. 1 or 2 non-polypeptide moietiesattached to the polypeptide. It will be understood that the conjugatealso exhibits an interferon-alpha activity (such as, antiviral activity,T_(H)1 differentiation activity, and/or antiproliferative activity).

In another aspect the conjugate comprises a polypeptide exhibiting aninterferon-alpha activity which comprises an amino acid sequence whichdiffers in 1 to 16 amino acid positions from SEQ ID NO:1 and comprisesone or more substitutions relative to SEQ ID NO:1 selected from thegroup consisting of F48A/L; V51P; F55A; F65A; F68P; L111A; and V114P;and at least one non-polypeptide moiety attached to the polypeptide,such as e.g., 1-6, 1-5, 1-4, 1-3, e.g. 1 or 2 non-polypeptide moietiesattached to the polypeptide. In some instances, the amino acid sequencefurther comprises one or more substitutions which introduces anattachment group for the non-polypeptide moiety (e.g., by substitutionof an amino acid residue for a different residue which comprises anattachment group for the non-polypeptide moiety, or by insertion of anadditional amino acid residue which comprises an attachment group forthe non-polypeptide moiety). It will be understood that the conjugatealso exhibits an interferon-alpha activity (such as, antiviral activity,T_(H)1 differentiation activity, and/or antiproliferative activity). Theinvention also provides conjugates which each comprise a polypeptidecomprising a sequence which (a) differs in 1 to 16 amino acid positionsfrom SEQ ID NO:1 and (b) comprises the substitution F48A/L relative toSEQ ID NO:1, and at least one non-polypeptide moiety attached to thepolypeptide, such as e.g., 1-6, 1-5, 1-4, 1-3, e.g. 1 or 2non-polypeptide moieties attached to the polypeptide. Some suchconjugates comprise a polypeptide sequence which differs from SEQ IDNO:1 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 aminoacid positions, e.g., in 1-15 amino acid positions, in 1-14 amino acidpositions, in 1-13 amino acid positions, in 1-12 amino acid positions,1-11 amino acid positions, in 1-10 amino acid positions, in 1-9 aminoacid positions, in 1-8 amino acid positions, in 1-7 amino acidpositions, in 1-6 amino acid positions, in 1-5 amino acid positions, in1-4 amino acid positions, in 1-3 amino acid positions, or in 1-2 aminoacid positions. Some such conjugates comprise the substitution F48Arelative to SEQ ID NO:1. In some instances, the polypeptide part of theconjugate further comprises one or more substitution(s) relative to SEQID NO:1 selected from the group consisting of H47Q; V51A/P; Q52P/E;A53T; F55A/S; L56V; F65A; F57L; Y58H; M61I; F68P; L111A; N113K; V114E/P;and E160D. Some such conjugates exhibit an interferon-alpha activity(e.g., antiviral activity, T_(H)1 differentiation activity, and/orantiproliferative activity). Some such conjugates further comprise oneor more additional amino acid(s), such as a methionine added to theN-terminus of the polypeptide.

The invention also provides conjugates which each comprise a polypeptidecomprising a sequence which (a) differs in 1 to 16 amino acid positionsfrom SEQ ID NO:1 and (b) comprises the substitution F55A relative to SEQID NO:1, and at least one non-polypeptide moiety attached to thepolypeptide, such as e.g., 1-6, 1-5, 1-4, 1-3, e.g. 1 or 2non-polypeptide moieties attached to the polypeptide. Some suchconjugates comprise a polypeptide sequence which differs from SEQ IDNO:1 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 aminoacid positions, e.g., in 1-15 amino acid positions, in 1-14 amino acidpositions, in 1-13 amino acid positions, in 1-12 amino acid positions,1-11 amino acid positions, in 1-10 amino acid positions, in 1-9 aminoacid positions, in 1-8 amino acid positions, in 1-7 amino acidpositions, in 1-6 amino acid positions, in 1-5 amino acid positions, in1-4 amino acid positions, in 1-3 amino acid positions, or in 1-2 aminoacid positions. In some instances, the polypeptide part of the conjugatefurther comprises one or more substitution(s) relative to SEQ ID NO:1selected from the group consisting of H47Q; F48A/L; V51A/P; Q52P/E;A53T; L56V; F65A; F57L; Y58H; M61I; F68P; L111A; N113K; V114E/P; andE160D. Some such conjugates exhibit an interferon-alpha activity (e.g.,antiviral activity, T_(H)1 differentiation activity, and/orantiproliferative activity). Some such conjugates further comprise oneor more additional amino acid(s), such as a methionine added to theN-terminus of the polypeptide.

The invention also provides conjugates which each comprise a polypeptidecomprising a sequence which (a) differs in 1 to 16 amino acid positionsfrom SEQ ID NO:1 and (b) comprises the substitution F65A relative to SEQID NO:1, and at least one non-polypeptide moiety attached to thepolypeptide, such as e.g., 1-6, 1-5, 1-4, 1-3, e.g. 1 or 2non-polypeptide moieties attached to the polypeptide. Some suchconjugates comprise a polypeptide sequence which differs from SEQ IDNO:1 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 aminoacid positions, e.g., in 1-15 amino acid positions, in 1-14 amino acidpositions, in 1-13 amino acid positions, in 1-12 amino acid positions,1-11 amino acid positions, in 1-10 amino acid positions, in 1-9 aminoacid positions, in 1-8 amino acid positions, in 1-7 amino acidpositions, in 1-6 amino acid positions, in 1-5 amino acid positions, in1-4 amino acid positions, in 1-3 amino acid positions, or in 1-2 aminoacid positions. In some instances, the polypeptide part of the conjugatefurther comprises one or more substitutions relative to SEQ ID NO:1selected from the group consisting of H47Q; F48A/L; V51A/P; Q52P/E;A53T; F55A/S; L56V; F57L; Y58H; M611; F68P; L111A; N113K; V114E/P; andE160D. Some such conjugates exhibit an interferon-alpha activity (e.g.,antiviral activity, T_(H)1 differentiation activity, and/orantiproliferative activity). Some such conjugates further comprise oneor more additional amino acid(s), such as a methionine added to theN-terminus of the polypeptide.

The invention also provides conjugates which each comprise a polypeptidecomprising a sequence which (a) differs in 1 to 16 amino acid positionsfrom SEQ ID NO:1 and (b) comprises the substitution L111A relative toSEQ ID NO:1, and at least one non-polypeptide moiety attached to thepolypeptide, such as e.g., 1-6, 1-5, 1-4, 1-3, e.g. 1 or 2non-polypeptide moieties attached to the polypeptide. Some suchconjugates comprise a polypeptide sequence which differs from SEQ IDNO:1 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 aminoacid positions, e.g., in 1-15 amino acid positions, in 1-14 amino acidpositions, in 1-13 amino acid positions, in 1-12 amino acid positions,1-11 amino acid positions, in 1-10 amino acid positions, in 1-9 aminoacid positions, in 1-8 amino acid positions, in 1-7 amino acidpositions, in 1-6 amino acid positions, in 1-5 amino acid positions, in1-4 amino acid positions, in 1-3 amino acid positions, or in 1-2 aminoacid positions. In some instances, the polypeptide part of the conjugatefurther comprises one or more substitutions relative to SEQ ID NO:1selected from the group consisting of H47Q; F48A/L; V51A/P; Q52P/E;A53T; F55A/S; L56V; F65A; F57L; Y58H; M61I; F68P; N113K; V114E/P; andE160D. Some such conjugates exhibit an interferon-alpha activity (e.g.,antiviral activity, T_(H)1 differentiation activity, and/orantiproliferative activity). Some such conjugates further comprise oneor more additional amino acid(s), such as a methionine added to theN-terminus of the polypeptide.

The invention also provides conjugates which each comprise a polypeptidecomprising a sequence which (a) differs in 0 to 16 amino acid positionsfrom SEQ ID NO:13 and (b) comprises one or more of: Ala or Leu atposition 48; Pro at position 51; Ala at position 55; Ala at position 65;Pro at position 68; Ala at position 111; and Pro at position 114(position numbering relative to SEQ ID NO:13), and at least onenon-polypeptide moiety attached to the polypeptide, such as e.g., 1-6,1-5, 1-4, 1-3, e.g. 1 or 2 non-polypeptide moieties attached to thepolypeptide. Some such conjugates comprise a polypeptide comprising asequence which differs from SEQ ID NO:13 in 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 16 amino acid positions, e.g., in 0-15amino acid positions, in 0-14 amino acid positions, in 0-13 amino acidpositions, in 0-12 amino acid positions, in 0-11 amino acid positions,in 0-10 amino acid positions, in 0-9 amino acid positions, in 0-8 aminoacid positions, in 0-7 amino acid positions, in 0-6 amino acidpositions, in 0-5 amino acid positions, in 0-4 amino acid positions, in0-3 amino acid positions, in 0-2 amino acid positions, or in 0-1 aminoacid positions. Some such conjugates comprise a polypeptide sequencewhich comprises an Ala or a Leu at position 48. Some such conjugatescomprise a polypeptide sequence which comprises an Ala at position 48.In some instances, the polypeptide part of the conjugate furthercomprises one or more substitution(s) relative to SEQ ID NO:13 selectedfrom the group consisting of H47Q; V51A; Q52P/E; A53T; F55S; L56V; F57L;Y58H; M61I; N113K; V114E; and E160D. Some such conjugates exhibit aninterferon-alpha activity (e.g., antiviral activity, T_(H)1differentiation activity, and/or antiproliferative activity). Some suchconjugates comprise a polypeptide sequence further comprising one ormore additional amino acid(s), such as a methionine added to theN-terminus.

In another aspect the conjugate comprises a polypeptide exhibiting aninterferon-alpha activity which comprises an amino acid sequence whichdiffers in 1 to 16 amino acid positions from SEQ ID NO:36 and comprisesone or more substitutions relative to SEQ ID NO:36 selected from thegroup consisting of M21A; 124P; F48A/L; T51P; S55A; F65A; F68P; F90A;M93P; L111A; V114P; F124A; 1127P; and E160D; and at least onenon-polypeptide moiety attached to the polypeptide, such as e.g., 1-6,1-5, 1-4, 1-3, e.g. 1 or 2 non-polypeptide moieties attached to thepolypeptide. In some instances, the amino acid sequence furthercomprises one or more substitutions which introduces an attachment groupfor the non-polypeptide moiety (e.g., by substitution of an amino acidresidue for a different residue which comprises an attachment group forthe non-polypeptide moiety, or by insertion of an additional amino acidresidue which comprises an attachment group for the non-polypeptidemoiety).

The invention also provides conjugates which each comprise a polypeptidecomprising a sequence which (a) differs in 1 to 16 amino acid positionsfrom SEQ ID NO:36 and (b) comprises the substitution F48A/L relative toSEQ ID NO:36, and at least one non-polypeptide moiety attached to thepolypeptide, such as e.g., 1-6, 1-5, 1-4, 1-3, e.g. 1 or 2non-polypeptide moieties attached to the polypeptide. Some suchconjugates comprise a polypeptide sequence which differs from SEQ IDNO:36 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 aminoacid positions, e.g., in 1-15 amino acid positions, in 1-14 amino acidpositions, in 1-13 amino acid positions, in 1-12 amino acid positions,1-11 amino acid positions, in 1-10 amino acid positions, in 1-9 aminoacid positions, in 1-8 amino acid positions, in 1-7 amino acidpositions, in 1-6 amino acid positions, in 1-5 amino acid positions, in1-4 amino acid positions, in 1-3 amino acid positions, or in 1-2 aminoacid positions. Some such conjugates comprise the substitution F48Arelative to SEQ ID NO:36. In some instances, the polypeptide part of theconjugate further comprises one or more substitution(s) relative to SEQID NO:36 selected from the group consisting of M21A; I24P; P26L; H47Q;T51V/P; S55P/F/A; V56L; H58Y; L60M; F65A; F68P; F90Y/A; M93L/P; N95D;L111A; N113K; V114E/P; F124A; R125Q; I127P; T132K; L154F; and E160D.Some such conjugates exhibit an interferon-alpha activity (e.g.,antiviral activity, T_(H)1 differentiation activity, and/orantiproliferative activity). Some such conjugates further comprise oneor more additional amino acid(s), such as a methionine added to theN-terminus of the polypeptide.

The invention also provides conjugates which each comprise a polypeptidecomprising a sequence which (a) differs in 1 to 16 amino acid positionsfrom SEQ ID NO:36 and (b) comprises the substitution F90A relative toSEQ ID NO:36, and at least one non-polypeptide moiety attached to thepolypeptide, such as e.g., 1-6, 1-5, 1-4, 1-3, e.g. 1 or 2non-polypeptide moieties attached to the polypeptide. Some suchconjugates comprise a polypeptide sequence which differs from SEQ IDNO:36 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 aminoacid positions, e.g., in 1-15 amino acid positions, in 1-14 amino acidpositions, in 1-13 amino acid positions, in 1-12 amino acid positions,1-11 amino acid positions, in 1-10 amino acid positions, in 1-9 aminoacid positions, in 1-8 amino acid positions, in 1-7 amino acidpositions, in 1-6 amino acid positions, in 1-5 amino acid positions, in1-4 amino acid positions, in 1-3 amino acid positions, or in 1-2 aminoacid positions. In some instances, the polypeptide part of the conjugatefurther comprises one or more substitution(s) relative to SEQ ID NO:36selected from the group consisting of M21A; I24P; P26L; H47Q; F48A/L;T51V/P; S55P/F/A; V56L; H58Y; L60M; F65A; F68P; M93L/P; N95D; L111A;N113K; Vi14E/P; F124A; R125Q; I127P; T132K; L154F; and E160D. Some suchconjugates exhibit an interferon-alpha activity (e.g., antiviralactivity, T_(H)1 differentiation activity, and/or antiproliferativeactivity). Some such conjugates further comprise one or more additionalamino acid(s), such as a methionine added to the N-terminus of thepolypeptide.

The invention also provides conjugates which each comprise a polypeptidecomprising a sequence which (a) differs in 1 to 16 amino acid positionsfrom SEQ ID NO:36 and (b) comprises the substitution E160D relative toSEQ ID NO:36, and at least one non-polypeptide moiety attached to thepolypeptide, such as e.g., 1-6, 1-5, 1-4, 1-3, e.g. 1 or 2non-polypeptide moieties attached to the polypeptide. Some suchconjugates comprise a polypeptide sequence which differs from SEQ IDNO:36 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 aminoacid positions, e.g., in 1-15 amino acid positions, in 1-14 amino acidpositions, in 1-13 amino acid positions, in 1-12 amino acid positions,1-11 amino acid positions, in 1-10 amino acid positions, in 1-9 aminoacid positions, in 1-8 amino acid positions, in 1-7 amino acidpositions, in 1-6 amino acid positions, in 1-5 amino acid positions, in1-4 amino acid positions, in 1-3 amino acid positions, or in 1-2 aminoacid positions. In some instances, the polypeptide part of the conjugatefurther comprises one or more substitution(s) relative to SEQ ID NO:36selected from the group consisting of M21A; I24P; P26L; H47Q; F48A/L;T51V/P; S55P/F/A; V56L; H58Y; L60M; F65A; F68P; F90Y/A; M93L/P; N95D;L111A; N113K; V114E/P; F124A; R125Q; I127P; T132K; and L154F. Some suchconjugates exhibit an interferon-alpha activity (e.g., antiviralactivity, T_(H)1 differentiation activity, and/or antiproliferativeactivity). Some such conjugates further comprise one or more additionalamino acid(s), such as a methionine added to the N-terminus of thepolypeptide.

The invention also provides conjugates which each comprise a polypeptidecomprising a sequence which (a) differs in 0 to 16 amino acid positionsfrom SEQ ID NO:38 and (b) comprises one or more of: Ala at position 21;Pro at position 24; Ala or Leu at position 48; Pro at position 51; Alaat position 55; Ala at position 65; Pro at position 68; Ala at position90; Pro at position 93; Ala at position 111; Pro at position 114; Ala atposition 124; Pro at position 127; and Glu at position 160 (positionnumbering relative to SEQ ID NO:38), and at least one non-polypeptidemoiety attached to the polypeptide, such as e.g., 1-6, 1-5, 1-4, 1-3,e.g. 1 or 2 non-polypeptide moieties attached to the polypeptide. Somesuch conjugates comprise a polypeptide comprising a sequence whichdiffers from SEQ ID NO:38 in 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, or 16 amino acid positions, e.g., in 0-15 amino acidpositions, in 0-14 amino acid positions, in 0-13 amino acid positions,in 0-12 amino acid positions, in 0-11 amino acid positions, in 0-10amino acid positions, in 0-9 amino acid positions, in 0-8 amino acidpositions, in 0-7 amino acid positions, in 0-6 amino acid positions, in0-5 amino acid positions, in 0-4 amino acid positions, in 0-3 amino acidpositions, in 0-2 amino acid positions, or in 0-1 amino acid positions.Some such conjugates comprise a polypeptide sequence which comprises anAla or a Leu at position 48. Some such conjugates comprise a polypeptidesequence which comprises an Ala at position 48. In some instances, thepolypeptide part of the conjugate further comprises one or moresubstitution(s) relative to SEQ ID NO:38 selected from the groupconsisting of P26L; H47Q; V51T; F55P/S; L56V; Y58H; L60M; F90Y; M93L;N95D; N113K; V114E; R125Q; T132K; and F154L. Some such conjugatesexhibit an interferon-alpha activity (e.g., antiviral activity, T_(H)1differentiation activity, and/or antiproliferative activity). Some suchconjugates comprise a polypeptide sequence further comprising one ormore additional amino acid(s), such as a methionine added to theN-terminus.

The term “conjugate” (or interchangeably “polypeptide conjugate” or“conjugated polypeptide”) is intended to indicate a heterogeneous (inthe sense of composite) molecule formed by the covalent attachment ofone or more polypeptides of the invention to one or more non-polypeptidemoieties. The term “covalent attachment” means that the polypeptide andthe non-polypeptide moiety are either directly covalently joined to oneanother, or else are indirectly covalently joined to one another throughan intervening moiety or moieties, such as a bridge, spacer, or linkagemoiety or moieties. Preferably, a conjugated polypeptide is soluble atrelevant concentrations and conditions, i.e. soluble in physiologicalfluids such as blood. Examples of conjugated polypeptides of theinvention include glycosylated and/or PEGylated polypeptides. The term“non-conjugated polypeptide” may be used to refer to the polypeptidepart of the conjugated polypeptide.

The term “non-polypeptide moiety” is intended to mean a molecule that iscapable of conjugating to an attachment group of the polypeptide.Preferred examples of non-polypeptide moieties include polymermolecules, sugar moieties, lipophilic compounds, or organic derivatizingagents, in particular polymer molecules or sugar moieties. It will beunderstood that the non-polypeptide moiety is linked to the polypeptidethrough an attachment group of the polypeptide. Except where the numberof non-polypeptide moieties, such as polymer molecule(s), attached tothe polypeptide is expressly indicated, every reference to “anon-polypeptide moiety” attached to the polypeptide or otherwise used inthe present invention shall be a reference to one or morenon-polypeptide moieties attached to the polypeptide.

The term “polymer molecule” is defined as a molecule formed by covalentlinkage of two or more monomers, wherein none of the monomers is anamino acid residue. The term “polymer” may be used interchangeably withthe term “polymer molecule”.

The term “sugar moiety” is intended to indicate a carbohydrate moleculeattached by in vivo or in vitro glycosylation, such as N- orO-glycosylation.

An “N-glycosylation site” has the sequence N-X-S/T/C, wherein X is anyamino acid residue except proline, N is asparagine and S/T/C is eitherserine, threonine or cysteine, preferably serine or threonine, and mostpreferably threonine.

An “O-glycosylation site” comprises the OH-group of a serine orthreonine residue.

The term “attachment group” is intended to indicate an amino acidresidue group capable of coupling to the relevant non-polypeptide moietysuch as a polymer molecule or a sugar moiety. Non-limiting examples ofuseful attachment groups and some corresponding non-polypeptide moietiesare provided in Table 4 below. TABLE 4 Useful attachment groups andexamples of corresponding non-polypeptide moieties Examples ofconjugation Attachment Example of non- method/activated group Amino acidpolypeptide moieties PEG Reference —NH₂ N-terminus, Lys Polymer, e.g.PEG mPEG-SPA Nektar Inc. 2003 mPEG2-NHS Catalog mPEG2- butryALD —COOHC-terminus, Asp, Polymer, e.g. PEG mPEG-Hz Nektar Inc. 2003 Glu Sugarmoiety In vitro coupling Catalog —SH Cys Polymer, e.g. PEG, mPEG-VSNektar Inc. 2003 Sugar moiety mPEG2-MAL Catalog; In vitro couplingDelgado et al, Critical Reviews in Therapeutic Drug Carrier Systems9(3,4): 249-304 (1992) —OH Ser, Thr, OH— Sugar moiety In vivo O-linkedglycosylation —CONH₂ Asn as part of an Sugar moiety In vivo N-N-glycosylation glycosylation site Aromatic Phe, Tyr, Trp Sugar moietyIn vitro coupling residue —CONH₂ Gln Sugar moiety In vitro coupling Yanand Wold, Biochemistry, 1984, Jul 31; 23(16): 3759-65 Aldehyde OxidizedPolymer, e.g. PEG, PEGylation Andresz et al., Ketone carbohydratePEG-hydrazide 1978, Makromol. Chem. 179: 301; WO 92/16555, WO 00/23114Guanidino Arg Sugar moiety In vitro coupling Lundblad and Noyes,Chemical Reagents for Protein Modification, CRC Press Inc. Boca Raton,FI Imidazole ring His Sugar moiety In vitro coupling As for guanidine

For in vivo N-glycosylation, the term “attachment group” is used in anunconventional way to indicate the amino acid residues constituting anN-glycosylation site (with the sequence N-X-S/T/C, wherein X is anyamino acid residue except proline, N is asparagine and S/T/C is eitherserine, threonine or cysteine, preferably serine or threonine, and mostpreferably threonine). Although the asparagine residue of theN-glycosylation site is the one to which the sugar moiety is attachedduring glycosylation, such attachment cannot be achieved unless theother amino acid residues of the N-glycosylation site is present.Accordingly, when the non-polypeptide moiety is a sugar moiety and theconjugation is to be achieved by N-glycosylation, the term “amino acidresidue comprising an attachment group for the non-polypeptide moiety”as used in connection with alterations of the amino acid sequence of thepolypeptide of the invention is to be understood as one, two or all ofthe amino acid residues constituting an N-glycosylation site is/are tobe altered in such a manner that either a functional N-glycosylationsite is introduced into the amino acid sequence, removed from saidsequence or a functional N-glycosylation site is retained in the aminoacid sequence (e.g. by substituting a serine residue, which alreadyconstitutes part of an N-glycosylation site, with a threonine residueand vice versa).

The term “introduce” (i.e., an “introduced” amino acid residue,“introduction” of an amino acid residue) is primarily intended to meansubstitution of an existing amino acid residue for another amino acidresidue, but may also mean insertion of an additional amino acidresidue.

The term “remove” (i.e., a “removed” amino acid residue, “removal” of anamino acid residue) is primarily intended to mean substitution of theamino acid residue to be removed for another amino acid residue, but mayalso mean deletion (without substitution) of the amino acid residue tobe removed.

The term “amino acid residue comprising an attachment group for thenon-polypeptide moiety” is intended to indicate that the amino acidresidue is one to which the non-polypeptide moiety binds (in the case ofan introduced amino acid residue) or would have bound (in the case of aremoved amino acid residue).

The term “functional in vivo half-life” is used in its normal meaning,i.e. the time at which 50% of the biological activity of the polypeptideis still present in the body/target organ, or the time at which theactivity of the polypeptide is 50% of the initial value. The functionalin vivo half-life may be determined in an experimental animal, such asrat, mice, rabbit, dog or monkey. Preferably, the functional in vivohalf-life is determined in a non-human primate, such as a monkey.Furthermore, the functional in vivo half-life may be determined for asample that has been administered intravenously or subcutaneously.

As an alternative to determining functional in vivo half-life, “serumhalf-life” may be determined, i.e. the time at which 50% of thepolypeptide circulates in the plasma or bloodstream prior to beingcleared. Determination of serum half-life is often more simple thandetermining the functional in vivo half-life and the magnitude of serumhalf-life is usually a good indication of the magnitude of functional invivo half-life. Alternatively terms to serum half-life include “plasmahalf-life”, “circulating half-life”, “serum clearance”, “plasmaclearance” and “clearance half-life”. The serum half-life may bedetermined as described above in connection with determination offunctional in vivo half-life.

The term “serum” is used in its normal meaning, i.e. as blood plasmawithout fibrinogen and other clotting factors.

The term “increased” as used about the functional in vivo half-life orserum half-life is used to indicate that the relevant half-life of theconjugate of the invention is statistically significantly increasedrelative to that of a reference molecule, such as a wild-typeinterferon-alpha, e.g., a human interferon-alpha, such as one of SEQ IDNO:45-SEQ ID NO:56 (or other huIFN-alpha sequences as described hereinand/or in Allen G. and Diaz M. O. (1996), supra), or the correspondingnon-conjugated polypeptide. Thus, interesting conjugates of theinvention include those which have an increased functional in vivohalf-life or an increased serum half-life as compared to a referencemolecule mentioned above.

The term “AUC_(sc)” or “Area Under the Curve when administeredsubcutaneously” is used in its normal meaning, i.e. as the area underthe interferon-alpha-activity-in-serum vs. time curve, where theconjugated molecule has been administered subcutaneously to anexperimental animal. Once the experimental interferon-alpha activitytime points have been determined, the AUC_(sc) may conveniently becalculated by a computer program, such as GraphPad Prism 3.01.

The term “increased” as used about the AUC_(sc) is used to indicate thatthe Area Under the Curve for a conjugate of the invention, whenadministered subcutaneously, is statistically significantly increasedrelative to that of a reference molecule, such as wild-typeinterferon-alpha, e.g., a human interferon-alpha, such as one of SEQ IDNO:45-SEQ ID NO:56 (or other huIFN-alpha sequences as described hereinand/or in Allen G. and Diaz M. O. (1996), supra), or the correspondingnon-conjugated polypeptide, when determined under comparable conditions.Evidently, the same amount of interferon-alpha activity should beadministered for the conjugate of the invention and the referencemolecule. Consequently, in order to make direct comparisons betweendifferent interferon-alpha molecules, the AUC_(sc) values shouldtypically be normalized, i.e. be expressed as AUC_(sc)/doseadministered.

The term “T_(max,sc)” is used about the time point in theinterferon-alpha-activity-in-serum vs. time curve where the highestinterferon-alpha activity in serum is observed.

It will be understood that while the examples and modifications to theparent polypeptide are generally provided herein in regards to thesequence SEQ ID NO:1, the disclosed modifications may also be made inequivalent amino acid positions of any of the other polypeptides of theinvention (including SEQ ID NOs:2-35 and 37-44 and variants thereof)described above.

By removing and/or introducing amino acid residues comprising anattachment group for the non-polypeptide moiety it is possible tospecifically adapt the polypeptide so as to make the molecule moresusceptible to conjugation to the non-polypeptide moiety of choice, tooptimize the conjugation pattern (e.g. to ensure an optimal distributionof non-polypeptide moieties on the surface of the interferon-alphamolecule and thereby, e.g., effectively shield epitopes and othersurface parts of the polypeptide without significantly impairing thefunction thereof). For instance, by introduction of attachment groups,the interferon-alpha polypeptide is altered in the content of thespecific amino acid residues to which the relevant non-polypeptidemoiety binds, whereby a more efficient, specific and/or extensiveconjugation is achieved. By removal of one or more attachment groups itis possible to avoid conjugation to the non-polypeptide moiety in partsof the polypeptide in which such conjugation is disadvantageous, e.g. toan amino acid residue located at or near a functional site of thepolypeptide (since conjugation at such a site may result in inactivationor reduced interferon-alpha activity of the resulting conjugate due toimpaired receptor recognition). Further, it may be advantageous toremove an attachment group located close to another attachment group.

It will be understood that the amino acid residue comprising anattachment group for a non-polypeptide moiety, whether it be removed orintroduced, is selected on the basis of the nature of thenon-polypeptide moiety and, in some instances, on the basis of theconjugation method to be used. For instance, when the non-polypeptidemoiety is a polymer molecule, such as a polyethylene glycol orpolyalkylene oxide derived molecule, amino acid residues capable offunctioning as an attachment group may be selected from the groupconsisting of cysteine, lysine (and/or the N-terminal amino group of thepolypeptide), aspartic acid, glutamic acid, histidine and arginine. Whenthe non-polypeptide moiety is a sugar moiety, the attachment group is anin vivo or in vitro N- or O-glycosylation site, preferably anN-glycosylation site.

In some instances, when an attachment group for a non-polypeptide moietyis to be introduced into or removed from the interferon-alphapolypeptide, the position of the interferon-alpha polypeptide to bemodified may be conveniently selected as follows:

The position to be modified may be located at the surface of theinterferon-alpha polypeptide, such as a position occupied by an aminoacid residue which has more than 25% of its side chain exposed to thesolvent, such as more than 50% of its side chain exposed to the solvent.Such positions have been identified on the basis of an analysis of a 3Dstructure of the human interferon-alpha 2a molecule as described in the“Materials and Methods” section herein.

In order to determine an optimal distribution of attachment groups, thedistance between amino acid residues located at the surface of theinterferon-alpha molecule was calculated on the basis of a 3D structureof an interferon-alpha polypeptide. More specifically, the distancebetween the CB's of the amino acid residues comprising such attachmentgroups, or the distance between the functional group (NZ for lysine, CGfor aspartic acid, CD for glutamic acid, SG for cysteine) of one and theCB of another amino acid residue comprising an attachment group weredetermined. In case of glycine, CA was used instead of CB. In theinterferon-α polypeptide part of a conjugate of the invention, any ofsaid distances may be more than 8 Å, such as more than 10 Å in order toavoid or reduce heterogeneous conjugation and to provide a uniformdistribution of attachment groups, e.g. with the aim of epitopeshielding.

Furthermore, in the interferon-alpha polypeptide part of a conjugate ofthe invention, in some instances attachment groups located at or nearthe receptor binding sites of interferon-alpha are removed, such as bysubstitution of the amino acid residue comprising such group. In someinstances, amino acid residues comprising an attachment group for anon-polypeptide moiety, such as cysteine or lysine, are often notintroduced at or near the receptor binding site of the interferon alphamolecule.

Another approach for modifying an interferon-alpha polypeptide is toshield and thereby modify or destroy or otherwise inactivate an epitopepresent in the parent interferon-alpha, by conjugation to anon-polypeptide moiety. Epitopes of interferon-alpha polypeptides may beidentified by use of methods known in the art, also known as epitopemapping, see e.g. Romagnoli et al., J. Biol Chem., 1999, 380(5):553-9,DeLisser HM, Methods Mol Biol, 1999, 96:11-20, Van de Water et al., ClinImmunol Immunopathol, 1997, 85(3):229-35, Saint-Remy J M, Toxicology,1997, 119(1):77-81, and Lane D P and Stephen C W, Curr Opin Immunol,1993, 5(2):268-71. One method is to establish a phage display libraryexpressing random oligopeptides of, e.g., 9 amino acid residues. IgG1antibodies from specific antisera towards human interferon-alpha arepurified by immunoprecipitation and the reactive phages are identifiedby immunoblotting. By sequencing the DNA of the purified reactivephages, the sequence of the oligopeptide can be determined followed bylocalization of the sequence on the 3D-structure of theinterferon-alpha. Alternatively, epitopes can be identified according tothe method described in U.S. Pat. No. 5,041,376. The thereby identifiedregion on the structure constitutes an epitope that then can be selectedas a target region for introduction of an attachment group for thenon-polypeptide moiety. Preferably, at least one epitope, such as two,three or four epitopes of interferon-alpha are shielded by anon-polypeptide moiety according to the present invention. Accordingly,in one aspect, the conjugate of the invention has at least one shieldedepitope as compared to a wild type human interferon-alpha, including anycommercially available interferon-alpha. This may be done byintroduction of an attachment group for a non-polypeptide moiety into aposition located in the vicinity of (i.e. within 4 amino acid residuesin the primary sequence or within about 10 Å in the tertiary sequence)of a given epitope. The 10 Å distance is measured between CB's (CA's incase of glycine). Such specific introductions are described in thefollowing sections.

In case of removal of an attachment group, the relevant amino acidresidue comprising such group and occupying a position as defined abovemay be substituted with a different amino acid residue that does notcomprise an attachment group for the non-polypeptide moiety in question,or may be deleted. Removal of an N-glycosylation group, may also beaccomplished by insertion or removal of an amino acid reside within themotif N-X-S/T/C.

In case of introduction of an attachment group, an amino acid residuecomprising such group is introduced into the position, such as bysubstitution of the amino acid residue occupying such position.

The exact number of attachment groups available for conjugation andpresent in the interferon-alpha polypeptide is dependent on the effectdesired to be achieved by conjugation. The effect to be obtained is,e.g., dependent on the nature and degree of conjugation (e.g. theidentity of the non-polypeptide moiety, the number of non-polypeptidemoieties desirable or possible to conjugate to the polypeptide, wherethey should be conjugated or where conjugation should be avoided, etc.).For instance, if reduced immunogenicity is desired, the number (andlocation of) attachment groups should be sufficient to shield most orall epitopes. This is normally obtained when a greater proportion of theinterferon-alpha polypeptide is shielded. Effective shielding ofepitopes is normally achieved when the total number of attachment groupsavailable for conjugation is in the range of 1-6 attachment groups,e.g., 1-5, such as in the range of 1-3, such as 1, 2, or 3 attachmentgroups.

Functional in vivo half-life is i.a. dependent on the molecular weightof the conjugate, and the number of attachment groups needed forproviding increased half-life thus depends on the molecular weight ofthe non-polypeptide moiety in question. Some such conjugates comprise1-6, e.g., 1-5, such as 1-3, e.g. 1, 2, or 3 non-polypeptide moietieseach having a MW of about 2-40 kDa, such as about 2 kDa, about 5 kDa,about 12 kDa, about 15 kDa, about 20 kDa, about 30 kDa, or about 40 kDa.

In the conjugate of the invention, some, most, or substantially allconjugatable attachment groups are occupied by the relevantnon-polypeptide moiety.

The conjugate of the invention may exhibit one or more of the followingimproved properties:

For example, the conjugate may exhibit a reduced immunogenicity ascompared to a human interferon-alpha (such as any of the polypeptidesdefined herein as SEQ ID NO:45-57, or any other huIFN-alpha describedherein and/or in Allen G. and Diaz M. O. (1996), supra) or as comparedto the corresponding non-conjugated polypeptide, e.g. a reduction of atleast 10%, such as a reduction of at least of 25%, such as a reductionof at least of 50%, e.g. a reduction of at least 75% compared to thenon-conjugated polypeptide or compared to a human interferon-alpha.

In another aspect the conjugate may exhibit a reduced reaction or noreaction with neutralizing antibodies from patients treated with a humaninterferon-alpha (such as any of the polypeptides defined herein as SEQID NO: 45-57, or any other huIFN-alpha described herein and/or in AllenG. and Diaz M. O. (1996), supra) or as compared to the correspondingnon-conjugated polypeptide, e.g. a reduction of neutralisation of atleast 10%, such as at least of 25%, such as of at least 50%, e.g., atleast 75%.

In another aspect of the invention the conjugate may exhibit anincreased functional in vivo half-life and/or increased serum half-lifeas compared to a reference molecule such as a human interferon-alpha(e.g. any of the polypeptides defined herein as SEQ ID NO:45-57 or anyother huIFN-alpha described herein and/or in Allen G. and Diaz M. O.(1996), supra) or as compared to the corresponding non-conjugatedpolypeptide. Particular preferred conjugates are such conjugates wherethe ratio between the functional in vivo half-life (or serum half-life)of said conjugate and the functional in vivo half-life (or serumhalf-life) of said reference molecule is at least 1.25, such as at least1.50, such as at least 1.75, such as at least 2, such as at least 3,such as at least 4, such as at least 5, such as at least 6, such as atleast 7, such as at least 8. As mentioned above, the half-life isconveniently determined in an experimental animal, such as rat ormonkey, and may be based on intravenously or subcutaneouslyadministration.

In a further aspect the conjugate may exhibit an increasedbioavailability as compared to a reference molecule such as a humaninterferon-alpha (e.g. any of the polypeptides defined herein as SEQ IDNO:45-57, or any other huIFN-alpha described herein and/or in Allen G.and Diaz M. O. (1996), supra) or the corresponding non-conjugatedpolypeptide. For example, the conjugate may exhibit an increasedAUC_(sc) as compared to a reference molecule such as a humaninterferon-alpha or the corresponding non-conjugated polypeptide. Thus,exemplary conjugates are such conjugates where the ratio between theAUC_(sc) of said conjugate and the AUC_(sc) of said reference moleculeis at least 1.25, such as at least 1.5, such as at least 2, such as atleast 3, such as at least 4, such as at least 5 or at least 6, such asat least 7, such as at least 8, such as at least 9 or at least 10, suchas at least 12, such as at least 14, e.g. at least 16, at least 18 or atleast 20 when administered subcutaneously, in particular whenadministered subcutaneously in an experimental animal such as rat ormonkey. Analogously, some conjugates of the invention are suchconjugates wherein the ratio between Tmax for said conjugate and Tmaxfor said reference molecule, such as a human interferon-alpha or thecorresponding non-conjugated polypeptide, is at least 1.2, such as atleast 1.4, e.g. at least 1.6, such as at least 1.8, such as at least 2,e.g. at least 2.5, such as at least 3, such as at least 4, e.g. at least5, such as at least 6, such as at least 7, e.g. at least 8, such as atleast 9, such as at least 10, when administered subcutaneously, inparticular when administered subcutaneously in an experimental animalsuch as rat or monkey.

In some instances, the magnitude of the antiviral activity of aconjugate of the invention may be reduced (e.g. by at least about 75%,at least about 50%, at least about 25%, at least about 10%) or increased(e.g. by at least about 10%) or is about equal (e.g. within about +/−10%or about +/−5%) to that of a human interferon-alpha (e.g. any of thepolypeptides identified herein as SEQ ID NO:45-57, or any otherhuIFN-alpha described herein and/or in Allen G. and Diaz M. O. (1996),supra) or to that of the corresponding non-conjugated polypeptide. Insome instances the degree of antiviral activity as compared toantiproliferative activity of a conjugate of the invention may vary, andthus be higher, lower or about equal to that of a human interferon-alphaor to that of the corresponding non-conjugated polypeptide.

Conjugate of the Invention where the Non-Polypeptide Moiety Binds to aLysine Residue or the N-Terminal Amine

In one aspect, the invention relates to a conjugate exhibiting aninterferon-alpha activity and comprising at least one non-polypeptidemoiety conjugated to at least one lysine residue and/or to theN-terminal amino group of an interferon-alpha polypeptide comprising asequence selected from SEQ ID NOs:2-35 and 37-44. Some such conjugatescomprise a non-polypeptide moiety attached to a lysine residue selectedfrom K31, K50, K71, K84, K122, K134, K135, and K165; such as, forexample, a lysine residue selected from K31, K122, K135, and K165. Somesuch conjugates comprise a non-polypeptide moiety attached to a lysineresidue selected from K31, K122, and K135. Some such conjugates comprisea non-polypeptide moiety attached to a lysine residue selected from K122and K135. Some such conjugates comprise a non-polypeptide moietyattached to a lysine residue selected from K31 and K122; or K31 andK135.

In another aspect, the invention relates to a conjugate exhibiting aninterferon-alpha activity and comprising at least one non-polypeptidemoiety conjugated to at least one lysine residue, or to the N-terminalamino group, of an interferon-alpha polypeptide comprising a sequencewhich (a) differs in 1 to 16 amino acid positions (such as in 1-15 aminoacid positions, in 1-14 amino acid positions, in 1-13 amino acidpositions, in 1-12 amino acid positions, 1-11 amino acid positions, in1-10 amino acid positions, in 1-9 amino acid positions, in 1-8 aminoacid positions, in 1-7 amino acid positions, in 1-6 amino acidpositions, in 1-5 amino acid positions, in 1-4 amino acid positions, in1-3 amino acid positions, or in 1-2 amino acid positions) from SEQ IDNO:1 and (b) comprises one or more substitutions relative to SEQ ID NO:1selected from the group consisting of F48A/L; V51P; F55A; F65A; F68P;L111A; and V114P. Some such conjugates comprise a polypeptide sequencefurther comprising one or more additional amino acid(s), such as amethionine added to the N-terminus. Some conjugates according to thisaspect comprise at least one removed lysine residue and/or at least oneremoved histidine residue, and/or at least one introduced lysineresidue.

The invention also provides a conjugate exhibiting an interferon-alphaactivity and comprising at least one non-polypeptide moiety conjugatedto at least one lysine residue, or to the N-terminal amino group, of aninterferon-alpha polypeptide comprising a sequence which (a) differs in0 to 16 amino acid positions from SEQ ID NO:13 (such as in 0-15 aminoacid positions, in 0-14 amino acid positions, in 0-13 amino acidpositions, in 0-12 amino acid positions, in 0-11 amino acid positions,in 0-10 amino acid positions, in 0-9 amino acid positions, in 0-8 aminoacid positions, in 0-7 amino acid positions, in 0-6 amino acidpositions, in 0-5 amino acid positions, in 0-4 amino acid positions, in0-3 amino acid positions, in 0-2 amino acid positions, or in 0-1 aminoacid positions) and (b) comprises one or more of: Ala or Leu at position48; Pro at position 51; Ala at position 55; Ala at position 65; Pro atposition 68; Ala at position 111; and Pro at position 114 (positionnumbering relative to SEQ ID NO:13). Some such conjugates comprise apolypeptide sequence which comprises an Ala or a Leu at position 48.Some such conjugates comprise a polypeptide sequence which comprises anAla at position 48. In some instances, the polypeptide part of theconjugate further comprises one or more substitution(s) relative to SEQID NO:13 selected from the group consisting of H47Q; V51A; Q52P/E; A53T;F55S; L56V; F57L; Y58H; M61I; N113K; V114E; and E160D. Some suchconjugates comprise a polypeptide sequence further comprising one ormore additional amino acid(s), such as a methionine added to theN-terminus. Some conjugates according to this aspect comprise at leastone removed lysine residue and/or at least one removed histidineresidue, and/or at least one introduced lysine residue.

In another aspect, the invention relates to a conjugate exhibiting aninterferon-alpha activity and comprising at least one non-polypeptidemoiety conjugated to at least one lysine residue, or to the N-terminalamino group, of an interferon-alpha polypeptide comprising a sequencewhich (a) differs in 1 to 16 amino acid positions (such as in 1-15 aminoacid positions, in 1-14 amino acid positions, in 1-13 amino acidpositions, in 1-12 amino acid positions, 1-11 amino acid positions, in1-10 amino acid positions, in 1-9 amino acid positions, in 1-8 aminoacid positions, in 1-7 amino acid positions, in 1-6 amino acidpositions, in 1-5 amino acid positions, in 1-4 amino acid positions, in1-3 amino acid positions, or in 1-2 amino acid positions) from SEQ IDNO:36 and (b) comprises one or more substitutions relative to SEQ IDNO:36 selected from the group consisting of M21A; I24P; F48A/L; T51P;S55A; F65A; F68P; F90A; M93P; L111A; V114P; F124A; I127P; and E160D.Some such conjugates comprise a polypeptide sequence further comprisingone or more additional amino acid(s), such as a methionine added to theN-terminus. Some conjugates according to this aspect comprise at leastone removed lysine residue and/or at least one removed histidineresidue, and/or at least one introduced lysine residue.

The invention also provides a conjugate exhibiting an interferon-alphaactivity and comprising at least one non-polypeptide moiety conjugatedto at least one lysine residue, or to the N-terminal amino group, of aninterferon-alpha polypeptide comprising a sequence which (a) differs in0 to 16 amino acid positions (such as in 0-15 amino acid positions, in0-14 amino acid positions, in 0-13 amino acid positions, in 0-12 aminoacid positions, in 0-11 amino acid positions, in 0-10 amino acidpositions, in 0-9 amino acid positions, in 0-8 amino acid positions, in0-7 amino acid positions, in 0-6 amino acid positions, in 0-5 amino acidpositions, in 0-4 amino acid positions, in 0-3 amino acid positions, in0-2 amino acid positions, or in 0-1 amino acid positions) from SEQ IDNO:38 and (b) comprises one or more of: Ala at position 21; Pro atposition 24; Ala or Leu at position 48; Pro at position 51; Ala atposition 55; Ala at position 65; Pro at position 68; Ala at position 90;Pro at position 93; Ala at position 111; Pro at position 114; Ala atposition 124; Pro at position 127; and Glu at position 160 (positionnumbering relative to SEQ ID NO:38). Some such conjugates comprise apolypeptide sequence which comprises an Ala or a Leu at position 48.Some such conjugates comprise a polypeptide sequence which comprises anAla at position 48. In some instances, the polypeptide part of theconjugate further comprises one or more substitution(s) relative to SEQID NO:38 selected from the group consisting of P26L; H47Q; V51T; F55P/S;L56V; Y58H; L60M; F90Y; M93L; N95D; N113K; V114E; R125Q; T132K; andF154L. Some such conjugates comprise a polypeptide sequence furthercomprising one or more additional amino acid(s), such as a methionineadded to the N-terminus. Some conjugates according to this aspectcomprise at least one removed lysine residue and/or at least one removedhistidine residue, and/or at least one introduced lysine residue.

Some conjugates of the invention comprise a polypeptide sequencecomprising a substitution of an amino acid residue for a different aminoacid residue, or a deletion of an amino acid residue, which removes oneor more lysines, such as, one or more of K31, K50, K71, K84, K122, K133,K134, K135, and/or K165 from a polypeptide of the invention such as, forexample, one of SEQ ID NOs:2-35 and 37-44. The one or more lysineresidue(s) to be removed may be substituted with any other amino acid,may be substituted with an Arg (R) or Gln (Q), or may be deleted.

In instances where amine-reactive conjugation chemistries are employed,it may be advantageous to avoid or to minimize the potential forconjugation to histidine residues. Therefore, some conjugates of theinvention comprise a polypeptide sequence comprising a substitution or adeletion which removes one or more histidines, e.g., H7, H11, H34,and/or H47 (relative to SEQ ID NO:1) from any polypeptide sequence ofthe invention such as, for example, one of one of SEQ ID NOs:2-35 and37-44. The one or more histidine residue(s) to be removed may besubstituted with any other amino acid, may be substituted with an Arg(R) or Gln (Q), or may be deleted. Some such conjugates comprise thesubstitutions H34Q; H47Q; or H34Q+H47Q.

Alternatively, or in addition, some conjugates of the invention comprisea polypeptide sequence comprising a modification which introduces alysine into a position that is occupied in the parent sequence (e.g.,one of SEQ ID NOs:2-35 and 37-44) by an amino acid residue that isexposed to the surface of the molecule, e.g., one that has at least 25%,such as at least 50% of its side chain exposed to the surface. Some suchconjugates comprise a polypeptide sequence comprising one or more of thefollowing substitutions, relative to SEQ ID NO:1, which introduces alysine residue into a position which is predicted to be exposed at thesurface of the molecule with more than a 25% fractional ASA: D2K, L3K,P4K, Q5K, T6K, H7K, S8K, L9K, G10K, R12K, R13K, M16K, A19K, Q20K, R22K,R23K, I24K, S25K, L26K, F27K, S28K, L30K, R33K, H34K, D35K, R37K, Q40K,E41K, E42K, D44K, N46K, H47K, Q49K, V51K, Q52K, E59K, Q62K, Q63K, N66K,S69K, T70K, D72K, S74K, A75K, D78K, E79K, T80K, L81K, E83K, I87K, F90K,Q91K, N94K, D95K, E97K, A98K, V100K, M101K, Q102K, E103K, V104K, G105K,E107K, E108K, T109K, P110K, L111K, M112K, N113K, V114K, D115K, L118K,R121K, Q125K, R126K, T128K, L129K, T132K, E133K, Y136K, S137K, P138K,A146K, M149K, R150K, S153K, F154K, N157K, Q159K, E160K, S161K, L162K,R163K, S164K, and E166K, said amino acid residue positions relative toSEQ ID NO:1.

Some such conjugates of the invention comprise a polypeptide sequencecomprising one or more of the following substitutions, relative to SEQID NO:1, which introduces a lysine residue into a position which ispredicted to be exposed at the surface of the molecule with more than a50% fractional ASA: D2K, L3K, P4K, Q5K, T6K, H7K, S8K, L9K, R12K, R13K,M16K, A19K, S25K, F27K, S28K, R33K, H34K, D35K, R37K, E41K, D44K, N46K,H47K, Q49K, N66K, A75K, D78K, E79K, T80K, E83K, I87K, F90K, Q91K, N94K,D95K, M101K, Q102K, E103K, G105K, E107K, E108K, T109K, P110K, L111K,V114K, D115K, L118K, R121K, Q125K, R126K, L129K, T132K, E133K, P138K,R150K, E160K, L162K, R163K, S164K and E166K, said amino acid residuepositions relative to SEQ ID NO:1.

It is to be understood that while the examples of modifications to theparent polypeptide are generally provided herein relative to thesequence SEQ ID NO:1 (or relative to some other specified sequence), thedisclosed modifications may also be made in equivalent amino acidpositions of the other polypeptides of the invention (including SEQ IDNOs:2-35 and 37-44 and variants thereof) described herein. Thus, as anexample, the substitution H47K relative to SEQ ID NO:1 is understood tocorrespond to Q47K in SEQ ID NO:37, and so on.

Non-polypeptide moieties contemplated for this aspect of the inventioninclude polymer molecules, such as any of the molecules mentioned in thesection entitled “Conjugation to a polymer molecule”, such as PEG ormPEG or mPEG2. The conjugation between the lysine-containing polypeptideand the polymer molecule may be achieved in any suitable manner, e.g. asdescribed in the section entitled “Conjugation to a polymer molecule”,e.g. in using a one step method or in the stepwise manner referred to insaid section. An exemplary method for PEGylating the interferon-alphapolypeptide is to covalently attach PEG to lysine residues usinglysine-reactive PEGs, such as described in Example 5 herein. A number ofhighly specific, lysine-reactive PEGs (such as for example, succinimidylpropionate (SPA), succinimidyl butanoate (SBA), N-hydroxylsuccinimide(NHS), and aldehyde (e.g., ButyrALD)) and different size linear orbranched PEGs (e.g., 2-40 kDa, such as 2 kDa, 5 kDa, 12 kDa, 15 kDa, 20kDa, 30 kDa, or 40 kDa) are commercially available, e.g. from NektarTherapeutics Inc., Huntsville, Ala., USA, or SunBio, Anyang City, SouthKorea.

In another aspect, the invention includes a composition comprising apopulation of conjugates wherein the majority of the conjugates of saidpopulation each contain a single non-polypeptide moiety (such as, asingle polymer molecule, e.g., a single PEG, such as a linear PEG or abranched PEG ) covalently attached to a single lysine residue orN-terminal amino group of the polypeptide. For example, a“monoconjugated” (such as, a “monoPEGylated”) composition of theinvention comprises one or more “positional isomers” of said conjugate,wherein each positional isomer contains a single non-polypeptide moiety(e.g., a single PEG molecule) covalently attached to a single lysineresidue of the polypeptide.

The invention includes a monoPEGylated composition comprising apopulation of conjugates, wherein the majority of the conjugates of saidpopulation are positional isomers each containing a single PEG molecule(such as, a linear or branched PEG, such as a 2 kDa, 5 kDa, 12 kDa, 15kDa, 20 kDa, 30 kDa, or 40 kDa mPEG or mPEG2 molecule) covalentlyattached to a single lysine residue of a polypeptide of the invention.

Some such positional isomers contain a single PEG molecule covalentlyattached to a lysine residue selected from K31, K50, K71, K84, K122,K134, K135, and K165. Some such positional isomers contain a single PEGmolecule covalently attached to a lysine residue selected from K31,K122, K135, and K165. Some such positional isomers contain a single PEGmolecule covalently attached to a lysine residue selected from K31,K122, and K135, such as, a single PEG molecule covalently attached to alysine reside selected from K31 and K122; K31 and K135; or K122 andK135.

Example 5 herein describes the preparation of monoPEGylated compositionsof the invention. A monoPEGylated composition of a polypeptide of theinvention identified herein as 14epi18 (SEQ ID NO:13) reacted with a 40kDa branched PEG (mPEG2-NHS) according to the procedure of Example 5contains predominantly two positional isomers, one with a single PEGmoiety covalently attached to Lys122 and another with a single PEGmoiety covalently attached to Lys135. Accordingly, the inventionincludes a composition (e.g. a monoPEGylated composition), saidcomposition comprising a polypeptide conjugate comprising the sequenceSEQ ID NO:13 wherein a PEG moiety is covalently attached to Lys122 orLys135. The invention also includes a composition (e.g. a monoPEGylatedcomposition), said composition comprising a polypeptide conjugatecomprising the sequence SEQ ID NO:13 wherein a 40 kDa PEG moiety (e.g.,a 40 kDa branched PEG moiety) is covalently attached to Lys122 orLys135.

A monoPEGylated composition of another exemplary polypeptide of theinvention, identified herein as 25epi19 (SEQ ID NO:38), when reactedwith a 40 kDa branched PEG (mPEG2-NHS) according to the procedure ofExample 5 predominantly contains three positional isomers, one with asingle PEG moiety covalently attached to Lys 31, another with a singlePEG moiety covalently attached to Lys122, and another with a single PEGmoiety covalently attached to Lys135. Accordingly, the inventionincludes a composition (e.g. a monoPEGylated composition), saidcomposition comprising a polypeptide conjugate comprising the sequenceSEQ ID NO:38 wherein a PEG moiety is covalently attached to Lys31,Lys122, or Lys135. The invention also includes a composition (e.g. amonoPEGylated composition), said composition comprising a polypeptideconjugate comprising the sequence SEQ ID NO:38 wherein a 40 kDa PEGmoiety (e.g., a 40 kDa branched PEG moiety) is covalently attached toLys31, Lys122, or Lys135.

Conjugate of the Invention where the Non-Polypeptide Moiety Binds to aCysteine Residue

In one aspect, the invention relates to a conjugate exhibiting aninterferon-alpha activity and comprising at least one non-polypeptidemoiety conjugated to at least one cysteine residue of aninterferon-alpha polypeptide comprising a sequence which (a) differs in1 to 16 amino acid positions (such as in 1-15 amino acid positions, in1-14 amino acid positions, in 1-13 amino acid positions, in 1-12 aminoacid positions, 1-11 amino acid positions, in 1-10 amino acid positions,in 1-9 amino acid positions, in 1-8 amino acid positions, in 1-7 aminoacid positions, in 1-6 amino acid positions, in 1-5 amino acidpositions, in 1-4 amino acid positions, in 1-3 amino acid positions, orin 1-2 amino acid positions) from SEQ ID NO:1 and (b) comprises one ormore substitutions relative to SEQ ID NO:1 selected from the groupconsisting of F48A/L; V51P; F55A; F65A; F68P; L111A; and V114P. Someconjugates according to this aspect comprise at least one introducedcysteine residue.

In another aspect, the invention relates to a conjugate exhibiting aninterferon-alpha activity and comprising at least one non-polypeptidemoiety conjugated to at least one cysteine residue of aninterferon-alpha polypeptide comprising a sequence which (a) differs in1 to 16 amino acid positions (such as in 1-15 amino acid positions, in1-14 amino acid positions, in 1-13 amino acid positions, in 1-12 aminoacid positions, 1-11 amino acid positions, in 1-10 amino acid positions,in 1-9 amino acid positions, in 1-8 amino acid positions, in 1-7 aminoacid positions, in 1-6 amino acid positions, in 1-5 amino acidpositions, in 1-4 amino acid positions, in 1-3 amino acid positions, orin 1-2 amino acid positions) from SEQ ID NO:36 and (b) comprises one ormore substitutions relative to SEQ ID NO:36 selected from the groupconsisting of M21A; I24P; F48A/L; T51P; S55A; F65A; F68P; F90A; M93P;L111A; V114P; F124A; I127P; and E160D. Some conjugates according to thisaspect comprise at least one introduced cysteine residue.

For example, some conjugates of the invention comprise a polypeptidesequence further comprising one or more of the following substitutions,relative to SEQ ID NO:1, which introduces a cysteine residue into aposition which is predicted to be exposed at the surface of the moleculewith more than a 25% fractional ASA: D2C, L3C, P4C, Q5C, T6C, H7C, S8C,L9C, G10C, R12C, R13C, M16C, A19C, Q20C, R22C, R23C, I24C, S25C, L26C,F27C, S28C, L30C, K31C, R33C, H34C, D35C, R37C, Q40C, E41C, E42C, D44C,N46C, H47C, Q49C, K50C, V51C, Q52C, E59C, Q62C, Q63C, N66C, S69C, T70C,K71C, D72C, S74C, A75C, D78C, E79C, T80C, L81C, E83C, K84C, F90C, Q91C,N94C, D95C, E97C, A98C, V100C, M101C, Q102C, E103C, V104C, G105C, E107C,E108C, T109C, P110C, L111C, M112C, N113C, V114C, D115C, L118C, R121C,K122C, Q125C, R126C, T128C, L129C, T132C, E133C, K134C, K135C, Y136C,S137C, P138C, A146C, M149C, R150C, S153C, F154C, N157C, Q159C, E160C,S161C, L162C, R163C, S164C, K165C and E166C, said amino acid residuepositions relative to SEQ ID NO:1.

Some such conjugates of the invention comprise a polypeptide sequencefurther comprising one or more of the following substitutions, relativeto SEQ ID NO:1, which introduces a cysteine residue into a positionwhich is predicted to be exposed at the surface of the molecule withmore than a 50% fractional ASA: D2C, L3C, P4C, Q5C, T6C, H7C, S8C, L9C,R12C, R13C, M16C, A19C, S25C, F27C, S28C, K31C, R33C, H34C, D35C, R37C,E41C, D44C, N46C, H47C, Q49C, K50C, N66C, K71C, A75C, D78C, E79C, T80C,E83C, K84C, I87C, F90C, Q91C, N94C, D95C, M101C, Q102C, E103C, G105C,E107C, E108C, T109C, P110C, L111C, V114C, D115C, L118C, R121C, K122C,Q125C, R126C, L129C, T132C, E133C, K135C, P138C, R150C, E160C, L162C,R163C, S164C, K165C and E166C, said amino acid residue positionsrelative to SEQ ID NO:1.

It is to be understood that while the examples of modifications to theparent polypeptide are generally provided herein relative to thesequence SEQ ID NO:1 (or relative to some other specified sequence), thedisclosed modifications may also be made in equivalent amino acidpositions of the other polypeptides of the invention (including SEQ IDNOs:2-35 and 37-44 and variants thereof) described herein. Thus, as anexample, the substitution H47C relative to SEQ ID NO:1 is understood tocorrespond to Q47C in SEQ ID NO:37, and so on.

In some instances, only a single cysteine residue is introduced in orderto avoid formation of disulfide bridges between two or more introducedcysteine residues.

In interferon alphas, disulfide bonds are formed between cysteines atpositions 1/99 and 29/139. The disulfide bond 29/139 is essential forbiological activity, while the 1/99 bond can be reduced withoutsignificantly affecting biological activity (Beilharz M. W. et al.(1986) J. Interferon Res. 6(6):677-685). Thus, in another aspect of theinvention one of C1 or C99 is removed, preferably by substitution, e.g.C1S or C99S, thereby leaving the other cysteine residue available forconjugation to a non-polypeptide moiety.

Non-polypeptide moieties contemplated in this aspect of the inventioninclude polymer molecules, such as any of the molecules mentioned in thesection entitled “Conjugation to a polymer molecule”, such as PEG ormPEG. The conjugation between the cysteine-containing polypeptide andthe polymer molecule may be achieved in any suitable manner, e.g. asdescribed in the section entitled “Conjugation to a polymer molecule”,e.g. in using a one step method or in the stepwise manner referred to insaid section. An exemplary method for PEGylating the interferon-alphapolypeptide is to covalently attach PEG to cysteine residues usingcysteine-reactive PEGs. A number of highly specific, cysteine-reactivePEGs with different groups (e.g. orthopyridyl-disulfide (OPSS),maleimide (MAL) and vinylsulfone (VS)) and different size linear orbranched PEGs (e.g., 2-40 kDa, such as 2 kDa, 5 kDa, 12 kDa, 15 kDa, 20kDa, 30 kDa, or 40 kDa) are commercially available, e.g. from NektarTherapeutics Inc., Huntsville, Ala., USA, or SunBio, Anyang City, SouthKorea.

Non-Polypeptide Moiety of the Conjugate of the Invention

As indicated above, the non-polypeptide moiety of the conjugate of theinvention is generally selected from the group consisting of a polymermolecule, a lipophilic compound, a sugar moiety (e.g., by way of in vivoN-glycosylation) and an organic derivatizing agent. All of these agentsmay confer desirable properties to the polypeptide part of theconjugate, such as reduced immunogenicity, increased functional in vivohalf-life, increased serum half-life, increased bioavailability and/orincreased AUC_(sc). The polypeptide part of the conjugate is oftenconjugated to only one type of non-polypeptide moiety, but may also beconjugated to two or more different types of non-polypeptide moieties,e.g. to a polymer molecule and a sugar moiety, etc. The conjugation totwo or more different non-polypeptide moieties may be donesimultaneously or sequentially. The choice of non-polypeptidemoiety/moieties, depends especially on the effect desired to be achievedby the conjugation. For instance, sugar moieties have been foundparticularly useful for reducing immunogenicity, whereas polymermolecules such as PEG are of particular use for increasing functional invivo half-life and/or serum half-life. Using a combination of a polymermolecule and a sugar moiety may enhance the reduction in immunogenicityand the increase in functional in vivo or serum half-life.

In the following sections “Conjugation to a lipophilic compound”,“Conjugation to a polymer molecule”, “Conjugation to a sugar moiety” and“Conjugation to an organic derivatizing agent” conjugation to specifictypes of non-polypeptide moieties is described.

Conjugation to a Lipophilic Compound

For conjugation to a lipophilic compound the following polypeptidegroups may function as attachment groups: the N-terminus or C-terminusof the polypeptide, the hydroxy groups of the amino acid residues Ser,Thr or Tyr, the ε-amino group of Lys, the SH group of Cys or thecarboxyl group of Asp and Glu. The polypeptide and the lipophiliccompound may be conjugated to each other either directly or by use of alinker. The lipophilic compound may be a natural compound such as asaturated or unsaturated fatty acid, a fatty acid diketone, a terpene, aprostaglandin, a vitamin, a carotenoid or steroid, or a syntheticcompound such as a carbon acid, an alcohol, an amine and sulphonic acidwith one or more alkyl, aryl, alkenyl or other multiple unsaturatedcompounds. The conjugation between the polypeptide and the lipophiliccompound, optionally through a linker may be done according to methodsknown in the art, e.g. as described by Bodanszky in Peptide Synthesis,John Wiley, New York, 1976 and in WO 96/12505.

Conjugation to a Polymer Molecule

The polymer molecule to be coupled to the polypeptide may be anysuitable polymer molecule, such as a natural or synthetic homo-polymeror heteropolymer, typically with a molecular weight in the range ofabout 300-100,000 Da, such as about 1000-50,000 Da, e.g. in the range ofabout 1000-40,000 Da. More particularly, the polymer molecule, such asPEG, in particular mPEG, will typically have a molecular weight of about2, 5, 10, 12, 15, 20, 30, 40 or 50 kDa, in particular a molecular weightof about 5 kDa, about 10 kDa, about 12 kDa, about 15 kDa, about 20 kDa,about 30 kDa or about 40 kDa. The PEG molecule may be branched (e.g.,mPEG2), or may be unbranched (i.e., linear).

When used about polymer molecules herein, the word “about” indicates anapproximate average molecular weight and reflects the fact that therewill normally be a certain molecular weight distribution in a givenpolymer preparation.

Examples of homo-polymers include a polyol (i.e. poly-OH), a polyamine(i.e. poly-NH₂) and a polycarboxylic acid (i.e. poly-COOH). Ahetero-polymer is a polymer which comprises one or more differentcoupling groups, such as a hydroxyl group and an amine group.

Examples of suitable polymer molecules include polymer moleculesselected from the group consisting of polyalkylene oxide (PAO),including polyalkylene glycol (PAG), such as polyethylene glycol (PEG)and polypropylene glycol (PPG), branched PEGs (PEG2), poly-vinyl alcohol(PVA), poly-carboxylate, poly-(vinylpyrolidone), polyethylene-co-maleicacid anhydride, polystyrene-co-malic acid anhydride, dextran includingcarboxymethyl-dextran, or any other biopolymer suitable for reducingimmunogenicity and/or increasing functional in vivo half-life and/orserum half-life. Generally, polyalkylene glycol-derived polymers arebiocompatible, non-toxic, non-antigenic, non-immunogenic, have variouswater solubility properties, and are easily excreted from livingorganisms.

PEG is the preferred polymer molecule to be used, since it has only fewreactive groups capable of cross-linking compared to e.g.polysaccharides such as dextran. In particular, monofunctional PEG, e.g.monomethoxypolyethylene glycol (mPEG), is of interest since its couplingchemistry is relatively simple (only one reactive group is available forconjugating with attachment groups on the polypeptide). Consequently,the risk of cross-linking is eliminated, the resulting polypeptideconjugates are more homogeneous and the reaction of the polymermolecules with the polypeptide is easier to control.

To effect covalent attachment of the polymer molecule(s) to thepolypeptide, the hydroxyl end groups of the polymer molecule must beprovided in activated form, i.e. with reactive functional groups(examples of which include primary amino groups, hydrazide (HZ), thiol,succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide(SSA), succinimidyl propionate (SPA), succinimidyl butanoate (SBA),succinimidyl carboxymethylate (SCM), benzotriazole carbonate (BTC),N-hydroxysuccinimide (NHS), aldehyde, nitrophenylcarbonate (NPC), andtresylate (TRES)). Suitably activated polymer molecules are commerciallyavailable, e.g. from Nektar Therapeutics, Inc., Huntsville, Ala., USA;PolyMASC Pharmaceuticals plc, UK; or SunBio Corporation, Anyang City,South Korea. Alternatively, the polymer molecules can be activated byconventional methods known in the art, e.g. as disclosed in WO 90/13540.Specific examples of activated linear or branched polymer moleculessuitable for use in the present invention are described in the NektarTherapeutics, Inc. 2003 Catalog (“Nektar Molecule Engineering:Polyethylene Glycol and Derivatives for Advanced Pegylation, Catalog2003”), incorporated by reference herein. Specific examples of activatedPEG polymers include the following linear PEGs: NHS-PEG, SPA-PEG,SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, SCM-PEG, NOR-PEG,BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG,OPSS-PEG, IODO-PEG, and MAL-PEG, and branched PEGs, such as PEG2-NHS,PEG2-MAL, and those disclosed in U.S. Pat. No. 5,932,462 and U.S. Pat.No. 5,643,575, both of which are incorporated herein by reference.Furthermore, the following publications, incorporated herein byreference, disclose useful polymer molecules and/or PEGylationchemistries: U.S. Pat. No. 5,824,778, U.S. Pat. No. 5,476,653, WO97/32607, EP 229,108, EP 402,378, U.S. Pat. No. 4,902,502, U.S. Pat. No.5,281,698, U.S. Pat. No. 5,122,614, U.S. Pat. No. 5,219,564, WO92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO94/28024, WO 95/00162, WO 95/11924, WO 95/13090, WO 95/33490, WO96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 439508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131, U.S. Pat. No.5,736,625, WO 98/05363, EP 809 996, U.S. Pat. No. 5,629,384, WO96/41813, WO 96/07670, U.S. Pat. No. 5,473,034, U.S. Pat. No. 5,516,673,EP 605 963, U.S. Pat. No. 5,382,657, EP 510 356, EP 400 472, EP 183 503and EP 154 316.

The conjugation of the polypeptide and the activated polymer moleculesis conducted by use of any conventional method, e.g. as described in thefollowing references (which also describe suitable methods foractivation of polymer molecules): Harris and Zalipsky, eds.,Poly(ethylene glycol) Chemistry and Biological Applications, AZC,Washington; R. F. Taylor, (1991), “Protein immobilisation. Fundamentaland applications”, Marcel Dekker, N.Y.; S. S. Wong, (1992), “Chemistryof Protein Conjugation and Crosslinking”, CRC Press, Boca Raton; G. T.Hermanson et al., (1993), “Immobilized Affinity Ligand Techniques”,Academic Press, N.Y.

For PEGylation of cysteine residues the polypeptide is usually treatedwith a reducing agent, such as dithiothreitol (DDT) prior to PEGylation.The reducing agent is subsequently removed by any conventional method,such as by desalting. Conjugation of PEG to a cysteine residue typicallytakes place in a suitable buffer at pH 6-9 at temperatures varying from4° C. to 25° C. for periods up to about 16 hours. Examples of activatedPEG polymers for coupling to cysteine residues include the followinglinear and branched PEGs: vinylsulfone-PEG (PEG-VS), such asvinylsulfone-mPEG (mPEG-VS); orthopyridyl-disulfide-PEG (PEG-OPSS), suchas orthopyridyl-disulfide-mPEG (mPEG-OPSS); and maleimide-PEG (PEG-MAL),such as maleimide-mPEG (mPEG-MAL) and branched maleimide-mPEG2(mPEG2-MAL).

Pegylation of lysines often employs PEG-N-hydroxylsuccinimide (e.g.,mPEG-NHS or mPEG2-NHS), or esters such as PEG succinimidyl propionate(e.g., mPEG-SPA) or PEG succinimidyl butanoate (e.g., mPEG-SBA). One ormore PEGs can be attached to a protein within 30 minutes at pH 8-9.5 atroom temperature if about equimolar amounts of PEG and protein aremixed. A molar ratio of PEG to protein amino groups of 1-5 to 1 willusually suffice. Increasing pH increases the rate of reaction, whilelowering pH reduces the rate of reaction. These highly reactive activeesters can couple at physiological pH, but less reactive derivativestypically require higher pH. Low temperatures may also be employed if alabile protein is being used. Under low temperature conditions, a longerreaction time may be used.

N-terminal PEGylation is facilitated by the difference between the pKavalues of the α-amino group of the N-terminal amino acid (˜7.6 to 8.0)and the ε-amino group of lysine (˜10). PEGylation of the N-terminalamino group often employs PEG-aldehydes (such as mPEG-propionaldehyde ormPEG-butylaldehyde), which are more selective for amines and thus areless likely to react with the imidazole group of histidine; in addition,PEG reagents used for lysine conjugation (such as mPEG-SPA, mPEG-SBA, ormPEG-NHS) may also be used for conjugation of the N-terminal amine.Conjugation of a PEG-aldehyde to the N-terminal amino group typicallytakes place in a suitable buffer (such as, 100 mM sodium acetate or 100mM sodium bisphosphate buffer with 20 mM sodium cyanoborohydride) atpH˜5.0 overnight at temperatures varying from about 4° C. to 25° C.Useful N-terminal PEGylation methods and chemistries are also describedin U.S. Pat. No. 5,985,265 and U.S. Pat. No. 6,077,939, bothincorporated herein by reference.

Typically, linear PEG or mPEG polymers will have a molecular weight ofabout 5 kDa, about 10 kDa, about 12 kDa, about 15 kDa, about 20 kDa, orabout 30 kDa. Branched PEG (PEG2 or mPEG2) polymers will typically havea molecular weight of about 10 kDa, about 20 kDa, or about 40 kDa. Insome instances, the higher-molecular weight branched PEG2 reagents, suchas 20 kDa or 40 kDa PEG2, including e.g. mPEG2-NHS for lysinePEGylation, mPEG2-MAL for cysteine PEGylation, or MPEG2-aldehyde forN-terminal PEGylation (all available from Nektar Therapeutics, Inc,Huntsville Ala.), may be used. The branched structure of the PEG2compound results in a relatively large molecular volume, so fewerattached molecules (or, one attached molecule) may impart the desiredcharacteristics of the PEGylated molecule.

The skilled person will be aware that the activation method and/orconjugation chemistry to be used depends on the attachment group(s) ofthe interferon-alpha polypeptide as well as the functional groups of thepolymer (e.g., being amino, hydroxyl, carboxyl, aldehyde or sulfhydryl).The PEGylation may be directed towards conjugation to all availableattachment groups on the polypeptide (i.e. such attachment groups thatare exposed at the surface of the polypeptide) or may be directedtowards specific attachment groups, e.g. cysteine residues, lysineresidues, or the N-terminal amino group.

Furthermore, the conjugation may be achieved in one step or in astepwise manner (e.g. as described in WO 99/55377).

In some instances, the polymer conjugation is performed under conditionsaiming at reacting as many of the available polymer attachment groups aspossible with polymer molecules. This is achieved by means of a suitablemolar excess of the polymer in relation to the polypeptide. Typicalmolar ratios of activated polymer molecules to polypeptide are up toabout 1000-1, such as up to about 200-1 or up to about 100-1. In somecases, the ratio may be somewhat lower, however, such as up to about50-1, 10-1 or 5-1. Also equimolar ratios may be used.

It is also contemplated according to the invention to couple the polymermolecules to the polypeptide through a linker. Suitable linkers are wellknown to the skilled person. A preferred example is cyanuric chloride(Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581; U.S. Pat.No. 4,179,337; Shafer et al., (1986), J. Polym. Sci. Polym. Chem. Ed.,24, 375-378).

Subsequent to the conjugation residual activated polymer molecules areblocked according to methods known in the art, e.g. by addition ofprimary amine to the reaction mixture, and the resulting inactivatedpolymer molecules removed by a suitable method.

Covalent in vitro coupling of a sugar moiety to amino acid residues ofinterferon-alpha may be used to modify or increase the number or profileof sugar substituents. Depending on the coupling mode used, thecarbohydrate(s) may be attached to a) arginine and histidine (Lundbladand Noyes, Chemical Reagents for Protein Modification, CRC Press Inc.Boca Raton, Fla.), b) free carboxyl groups (e.g. of the C-terminal aminoacid residue, asparagine or glutamine), c) free sulfhydryl groups suchas that of cysteine, d) free hydroxyl groups such as those of serine,threonine, tyrosine or hydroxyproline, e) aromatic residues such asthose of phenylalanine or tryptophan or f) the amide group of glutamine.These amino acid residues constitute examples of attachment groups for asugar moiety, which may be introduced and/or removed in theinterferon-alpha polypeptide. Suitable methods of in vitro coupling aredescribed in WO 87/05330 and in Aplin et al., CRC Crit Rev. Biochem.,pp. 259-306, 1981. The in vitro coupling of sugar moieties or PEG toprotein- and peptide-bound Gln-residues can also be carried out bytransglutaminases (TGases), e.g. as described by Sato et al., 1996Biochemistry 35, 13072-13080 or in EP 725145.

Coupling to a Sugar Moiety

In order to achieve in vivo glycosylation of an interferon-alphapolypeptide that has been modified by introduction of one or moreglycosylation sites, the nucleotide sequence encoding the polypeptidepart of the conjugate is inserted in a glycosylating, eukaryoticexpression host. The expression host cell may be selected from fungal(filamentous fungal or yeast), insect, mammalian animal cells, fromtransgenic plant cells or from transgenic animals. Furthermore, theglycosylation may be achieved in the human body when using a nucleotidesequence encoding the polypeptide part of a conjugate of the inventionor a polypeptide of the invention in gene therapy. In one aspect thehost cell is a mammalian cell, such as a CHO cell, a COS cell, a BHK orHEK cell, e.g. HEK293, or an insect cell, such as an SF9 cell, or ayeast cell, e.g. Saccharomyces cerevisiae, Pichia pastoris or any othersuitable glycosylating host, e.g. as described further below.Optionally, sugar moieties attached to the interferon-α polypeptide byin vivo glycosylation are further modified by use ofglycosyltransferases, e.g. using the GlycoAdvance™ technology marketedby Neose, Horsham, Pa., USA. Thereby, it is possible to, e.g., increasethe sialyation of the glycosylated interferon-alpha polypeptidefollowing expression and in vivo glycosylation by CHO cells.

Coupling to an Organic Derivatizing Agent

Covalent modification of the interferon-alpha polypeptide may beperformed by reacting (an) attachment group(s) of the polypeptide withan organic derivatizing agent. Suitable derivatizing agents and methodsare well known in the art. For example, cysteinyl residues most commonlyare reacted with α-haloacetates (and corresponding amines), such aschloroacetic acid or chloroacetamide, to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteinyl residues also are derivatizedby reaction with bromotrifluoroacetone, α-bromo-β-(4-imidozoyl)propionicacid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyldisulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide is also useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0. Lysinyl andamino terminal residues are reacted with succinic or other carboxylicacid anhydrides. Derivatization with these agents has the effect ofreversing the charge of the lysinyl residues. Other suitable reagentsfor derivatizing α-amino-containing residues include imidoesters such asmethyl picolinimidate; pyridoxal phosphate; pyridoxal;chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea;2,4-pentanedione; and transaminase-catalyzed reaction with glyoxylate.Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineguanidino group. Carboxyl side groups (aspartyl or glutamyl orC-terminal amino acid residue) are selectively modified by reaction withcarbodiimides (R—N═C═N—R′), where R and R′ are different alkyl groups,such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Blocking of a Functional Site

Since excessive polymer conjugation may lead to a loss of activity ofthe interferon-α polypeptide to which the polymer is conjugated, it maybe advantageous to remove attachment groups located at the functionalsite or to block the functional site prior to conjugation. These latterstrategies constitute further aspects of the invention (the firststrategy being exemplified further above, e.g. by removal of lysineresidues which may be located close to a functional site). Morespecifically, according to the second strategy the conjugation betweenthe interferon-alpha polypeptide and the non-polypeptide moiety isconducted under conditions where the functional site of the polypeptideis blocked by a helper molecule capable of binding to the functionalsite of the polypeptide. Preferably, the helper molecule is one whichspecifically recognizes a functional site of the polypeptide, such as areceptor, in particular the type I interferon receptor. Alternatively,the helper molecule may be an antibody, in particular a monoclonalantibody recognizing the interferon-alpha polypeptide. In particular,the helper molecule may be a neutralizing monoclonal antibody.

The polypeptide is allowed to interact with the helper molecule beforeeffecting conjugation. This ensures that the functional site of thepolypeptide is shielded or protected and consequently unavailable forderivatization by the non-polypeptide moiety such as a polymer.Following its elution from the helper molecule, the conjugate betweenthe non-polypeptide moiety and the polypeptide can be recovered with atleast a partially preserved functional site.

The subsequent conjugation of the polypeptide having a blockedfunctional site to a polymer, a lipophilic compound, an organicderivatizing agent or any other compound is conducted in the normal way,e.g. as described in the sections above entitled “Conjugation to . . .”.

Irrespective of the nature of the helper molecule to be used to shieldthe functional site of the polypeptide from conjugation, it is desirablethat the helper molecule is free from or comprises only a few attachmentgroups for the non-polypeptide moiety of choice in parts of the moleculewhere the conjugation to such groups would hamper the desorption of theconjugated polypeptide from the helper molecule. Hereby, selectiveconjugation to attachment groups present in non-shielded parts of thepolypeptide can be obtained and it is possible to reuse the helpermolecule for repeated cycles of conjugation. For instance, if thenon-polypeptide moiety is a polymer molecule such as PEG, which has theepsilon amino group of a lysine or N-terminal amino acid residue as anattachment group, it is desirable that the helper molecule issubstantially free from conjugatable epsilon amino groups, preferablyfree from any epsilon amino groups. Accordingly, in some instances thehelper molecule is a protein or peptide capable of binding to thefunctional site of the polypeptide, which protein or peptide is freefrom any conjugatable attachment groups for the non-polypeptide moietyof choice.

In a further aspect the helper molecule is first covalently linked to asolid phase such as column packing materials, for instance Sephadex oragarose beads, or a surface, e.g. reaction vessel. Subsequently, thepolypeptide is loaded onto the column material carrying the helpermolecule and conjugation carried out according to methods known in theart, e.g. as described in the sections above entitled “Conjugation to .. . ”. This procedure allows the polypeptide conjugate to be separatedfrom the helper molecule by elution. The polypeptide conjugate is elutedby conventional techniques under physico-chemical conditions that do notlead to a substantive degradation of the polypeptide conjugate. Thefluid phase containing the polypeptide conjugate is separated from thesolid phase to which the helper molecule remains covalently linked. Theseparation can be achieved in other ways: For instance, the helpermolecule may be derivatized with a second molecule (e.g. biotin) thatcan be recognized by a specific binder (e.g. streptavidin). The specificbinder may be linked to a solid phase thereby allowing the separation ofthe polypeptide conjugate from the helper molecule-second moleculecomplex through passage over a second helper-solid phase column whichwill retain, upon subsequent elution, the helper molecule-secondmolecule complex, but not the polypeptide conjugate. The polypeptideconjugate may be released from the helper molecule in any appropriatefashion. De-protection may be achieved by providing conditions in whichthe helper molecule dissociates from the functional site of theinterferon-α to which it is bound. For instance, a complex between anantibody to which a polymer is conjugated and an anti-idiotypic antibodycan be dissociated by adjusting the pH to an acid or alkaline pH.

Conjugation of a Tagged Interferon-Alpha Polypeptide

In another aspect the interferon-alpha polypeptide is expressed as afusion protein with a tag, i.e. an amino acid sequence or peptide madeup of typically 1-30, such as 1-20 or 1-15 or 1-10 or 1-5 amino acidresidues, e.g. added to the N-terminus or to the C-terminus of thepolypeptide. Besides allowing for fast and easy purification, the tag isa convenient tool for achieving conjugation between the taggedpolypeptide and the non-polypeptide moiety. In particular, the tag maybe used for achieving conjugation in microtiter plates or othercarriers, such as paramagnetic beads, to which the tagged polypeptidecan be immobilised via the tag. The conjugation to the taggedpolypeptide in, e.g., microtiter plates has the advantage that thetagged polypeptide can be immobilised in the microtiter plates directlyfrom the culture broth (in principle without any purification) andsubjected to conjugation. Thereby, the total number of process steps(from expression to conjugation) can be reduced. Furthermore, the tagmay function as a spacer molecule ensuring an improved accessibility tothe immobilised polypeptide to be conjugated. The conjugation using atagged polypeptide may be to any of the non-polypeptide moietiesdisclosed herein, e.g. to a polymer molecule such as PEG.

The identity of the specific tag to be used is not critical as long asthe tag is capable of being expressed with the polypeptide and iscapable of being immobilised on a suitable surface or carrier material.A number of suitable tags are commercially available, e.g. from UnizymeLaboratories, Denmark. Antibodies against such tags are commerciallyavailable, e.g. from ADI, Aves Lab and Research Diagnostics.

Polynucleotides of the Invention

The invention provides isolated or recombinant nucleic acids (alsoreferred to herein as polynucleotides), collectively referred to as“nucleic acids (or polynucleotides) of the invention”, which encodepolypeptides of the invention. The polynucleotides of the invention areuseful in a variety of applications. As discussed above, thepolynucleotides are useful in producing polypeptides of the invention.In addition, polynucleotides of the invention can be incorporated intoexpression vectors useful for gene therapy, DNA vaccination, andimmunotherapy, as described in more detail below.

In one aspect, the invention provides isolated or recombinantpolynucleotides that each comprise a nucleic acid sequence which encodesa polypeptide comprising an amino acid sequence selected from SEQ IDNOs:2-35 and 37-44; or, a complementary nucleic acid sequence thereof.Exemplary nucleic acid sequences according to this aspect of theinvention include, but are not limited to, SEQ ID NOs:59-61, which eachencode a polypeptide comprising the sequence SEQ ID NO:10; SEQ IDNOs:62-64 and 89, which each encode a polypeptide comprising thesequence SEQ ID NO:13; SEQ ID NOs:65-67, which each encode a polypeptidecomprising the sequence SEQ ID NO:15; SEQ ID NOs:68-70, which eachencode a polypeptide comprising the sequence SEQ ID NO:23; SEQ IDNOs:71-73, which each encode a polypeptide comprising the sequence SEQID NO:27; SEQ ID NOs:74-76, which each encode a polypeptide comprisingthe sequence SEQ ID NO:30; SEQ ID NOs:77-79, which each encode apolypeptide comprising the sequence SEQ ID NO:37; SEQ ID NOs:80-82 and90, which each encode a polypeptide comprising the sequence SEQ IDNO:38; SEQ ID NOs:83-85, which each encode a polypeptide comprising thesequence SEQ ID NO:41; and SEQ ID NOs:86-88, which each encode apolypeptide comprising the sequence SEQ ID NO:44.

The invention also provides isolated or recombinant polynucleotides thateach comprise a nucleic acid sequence which encodes a polypeptidecomprising an amino acid sequence which (a) differs in 1 to 16 aminoacid positions (such as in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, or 16 amino acid positions, e.g., in 1-15 amino acid positions,in 1-14 amino acid positions, in 1-13 amino acid positions, in 1-12amino acid positions, 1-11 amino acid positions, in 1-10 amino acidpositions, in 1-9 amino acid positions, in 1-8 amino acid positions, in1-7 amino acid positions, in 1-6 amino acid positions, in 1-5 amino acidpositions, in 1-4 amino acid positions, in 1-3 amino acid positions, orin 1-2 amino acid positions) from SEQ ID NO:1 and (b) comprises one ormore substitutions relative to SEQ ID NO:1 selected from the groupconsisting of F48A/L, V51P, F55A, F65A, F68P, L11A, and V114P; or, acomplementary nucleic acid sequence thereof. Some polypeptides encodedby polynucleotides of the invention further comprise one or moreadditional amino acid(s), such as a methionine added to the N-terminus.In some instances the encoded polypeptide exhibits an interferon-alphaactivity.

The invention also provides isolated or recombinant polynucleotides thateach comprise a nucleic acid sequence which encodes a polypeptidecomprising an amino acid sequence which (a) differs in 1 to 16 aminoacid positions (such as in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, or 16 amino acid positions, e.g., in 1-15 amino acid positions,in 1-14 amino acid positions, in 1-13 amino acid positions, in 1-12amino acid positions, 1-11 amino acid positions, in 1-10 amino acidpositions, in 1-9 amino acid positions, in 1-8 amino acid positions, in1-7 amino acid positions, in 1-6 amino acid positions, in 1-5 amino acidpositions, in 1-4 amino acid positions, in 1-3 amino acid positions, orin 1-2 amino acid positions) from SEQ ID NO:36 and (b) comprises one ormore substitutions relative to SEQ ID NO:36 selected from the groupconsisting of M21A, I24P, F48A/L, T51P, S55A, F65A, F68P, F90A, M93P,L111A, V114P, F124A, I127P, and E160D; or, a complementary nucleic acidsequence thereof. Some polypeptides encoded by polynucleotides of theinvention further comprise one or more additional amino acid(s), such asa methionine added to the N-terminus. In some instances the encodedpolypeptide exhibits an interferon-alpha activity.

The invention also provides isolated or recombinant polynucleotides thateach comprise a nucleic acid sequence which encodes a polypeptidecomprising an amino acid sequence having at least 90% sequence identity(e.g., at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% amino acid sequenceidentity) to any one of SEQ ID NOs:1-35, wherein the amino acid sequencecomprises one or more of: Ala or Leu at position 48; Pro at position 51;Ala at position 55; Ala at position 65; Pro at position 68; Ala atposition 111; and Pro at position 114, position numbering relative toSEQ ID NO:1; or, a complementary nucleic acid sequence thereof. Somepolypeptides encoded by polynucleotides of the invention furthercomprise one or more additional amino acid(s), such as a methionineadded to the N-terminus. In some instances the encoded polypeptideexhibits an interferon-alpha activity.

The invention also provides isolated or recombinant polynucleotides thateach comprise a nucleic acid sequence which encodes a polypeptidecomprising an amino acid sequence having at least 90% sequence identity(e.g., at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% amino acid sequenceidentity) to any one of SEQ ID NOs:36-44, wherein the amino acidsequence comprises one or more of: Ala at position 21; Pro at position24; Ala or Leu at position 48; Pro at position 51; Ala at position 55;Ala at position 65; Pro at position 68; Ala at position 90; Pro atposition 93; Ala at position 111; Pro at position 114; Ala at position124; Pro at position 127; and Asp at position 160, position numberingrelative to SEQ ID NO:36; or, a complementary nucleic acid sequencethereof. Some polypeptides encoded by polynucleotides of the inventionfurther comprise one or more additional amino acid(s), such as amethionine added to the N-terminus. In some instances the encodedpolypeptide exhibits an interferon-alpha activity.

Additional Aspects

Any of the polynucleotides of the invention (which includes thosedescribed above) may encode a fusion protein comprising at least oneadditional amino acid sequence, such as, for example, asecretion/localization sequence, a sequence useful for solubilization orimmobilization (e.g., for cell surface display) of the polypeptide, asequence useful for detection and/or purification of the polypeptide(e.g., a polypeptide purification subsequence, such as an epitope tag, apolyhistidine sequence, and the like). In another aspect, the inventionprovides cells comprising one or more of the polynucleotides of theinvention. Such cells may express one or more polypeptides encoded bythe polynucleotides of the invention.

The invention also provides vectors comprising any of thepolynucleotides of the invention. Such vectors may comprise a plasmid, acosmid, a phage, a virus, or a fragment of a virus. Such vectors maycomprise an expression vector, and, if desired, the nucleic acid isoperably linked to a promoter, including those discussed herein andbelow. Furthermore, in another aspect, the invention providescompositions comprising an excipient or carrier and at least one of anyof the polynucleotides of the invention, or vectors, cells, or hostcomprising such nucleic acids. Such composition may be pharmaceuticalcompositions, and the excipient or carrier may be a pharmaceuticallyacceptable excipient or carrier.

The invention also includes compositions comprising two or more nucleicacids of the invention, or fragments thereof (e.g., as substrates forrecombination). The composition can comprise a library of recombinantnucleic acids, where the library contains at least 2, at least 3, atleast 5, at least 10, at least 20, at least 50, or at least 100 or morenucleic acids described above. The nucleic acids are optionally clonedinto expression vectors, providing expression libraries.

The polynucleotides of the invention and fragments thereof, as well asvectors comprising such polynucleotides, may be employed for therapeuticor prophylactic uses in combination with a suitable carrier, such as apharmaceutical carrier. Such compositions comprise a therapeuticallyand/or prophylactically effective amount of the compound, and apharmaceutically acceptable carrier or excipient. Such a carrier orexcipient includes, but is not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Theformulation should suit the mode of administration. Methods ofadministering nucleic acids, polypeptides, and proteins are well knownin the art, and are further discussed below.

The invention also includes compositions produced by digesting one ormore of any of the nucleic acids of the invention with a restrictionendonuclease, an RNAse, or a DNAse (e.g., as is performed in certain ofthe recombination formats noted above); and compositions produced byfragmenting or shearing one or more nucleic acids of the invention bymechanical means (e.g., sonication, vortexing, and the like), which canalso be used to provide substrates for recombination in the methodsdescribed herein. The invention also provides compositions produced bycleaving at least one of any of the nucleic acids of the invention. Thecleaving may comprise mechanical, chemical, or enzymatic cleavage, andthe enzymatic cleavage may comprise cleavage with a restrictionendonuclease, an RNAse, or a DNAse.

Also included in the invention are compositions produced by a processcomprising incubating one or more of the fragmented nucleic acids of theinvention in the presence of ribonucleotide or deoxyribonucleotidetriphosphates and a nucleic acid polymerase. This resulting compositionforms a recombination mixture for many of the recombination formatsnoted above. The nucleic acid polymerase may be an RNA polymerase, a DNApolymerase, or an RNA-directed DNA polymerase (e.g., a “reversetranscriptase”); the polymerase can be, e.g., a thermostable DNApolymerase (e.g., VENT, TAQ, or the like).

Similarly, compositions comprising sets of oligonucleotidescorresponding to more than one nucleic acids of the invention are usefulas recombination substrates and are a feature of the invention. Forconvenience, these fragmented, sheared, or oligonucleotide synthesizedmixtures are referred to as fragmented nucleic acid sets.

The invention also provides an isolated or recombinant nucleic acidencoding a polypeptide that exhibits an interferon-alpha activity,produced by mutating or recombining at least one nucleic acid of theinvention.

Making Polynucleotides

Polynucleotides, oligonucleotides, and nucleic acid fragments of theinvention can be prepared by standard solid-phase methods, according toknown synthetic methods. Typically, fragments of up to about 100 basesare individually synthesized, then joined (e.g., by enzymatic orchemical ligation methods, or polymerase mediated recombination methods)to form essentially any desired continuous sequence. For example, thepolynucleotides and oligonucleotides of the invention can be prepared bychemical synthesis using, e.g., classical phosphoramidite methoddescribed by, e.g., Beaucage et al. (1981) Tetrahedron Letters22:1859-69, or the method described by Matthes et al. (1984) EMBO J3:801-05, e.g., as is typically practiced in automated syntheticmethods. According to the phosphoramidite method, oligonucleotides aresynthesized, e.g., in an automatic DNA synthesizer, purified, annealed,ligated and cloned into appropriate vectors.

In addition, essentially any polynucleotide can be custom ordered fromany of a variety of commercial sources, such as Operon Technologies Inc.(Alameda, Calif.) and many others. Similarly, peptides and antibodiescan be custom ordered from any of a variety of sources, e.g., CeltekPeptides (Nashville, Tenn.); Washington Biotechnology, Inc. (BaltimoreMd.); Global Peptide Services (Ft. Collin Co.), and many others.

Certain polynucleotides of the invention may also be obtained byscreening cDNA libraries (e.g., libraries generated by recombininghomologous nucleic acids as in typical recursive sequence recombinationmethods) using oligonucleotide probes that can hybridize to orPCR-amplify polynucleotides which encode interferon-alpha polypeptidesand fragments of those polypeptides. Procedures for screening andisolating cDNA clones are well-known to those of skill in the art. Suchtechniques are described in, e.g., Berger and Kimmel, Guide to MolecularCloning Techniques, Methods in Enzymol. Vol. 152, Acad. Press, Inc., SanDiego, Calif. (“Berger”); Sambrook, supra, and Current Protocols inMolecular Biology, Ausubel, supra. Some polynucleotides of the inventioncan be obtained by altering a naturally occurring sequence, e.g., bymutagenesis, recursive sequence recombination (e.g., shuffling), oroligonucleotide recombination. In other cases, such polynucleotides canbe made in silico or through oligonucleotide recombination methods asdescribed in the references cited herein.

As described in more detail herein, the polynucleotides of the inventioninclude polynucleotides that encode polypeptides of the invention,polynucleotide sequences complementary to these polynucleotidesequences, and polynucleotides that hybridize under at least stringentconditions to the sequences defined herein. A coding sequence refers toa polynucleotide sequence encoding a particular polypeptide or domain,region, or fragment of said polypeptide. A coding sequence may encode(code for) a polypeptide of the invention exhibiting an interferon alphaactivity as described above. The polynucleotides of the invention may bein the form of RNA or in the form of DNA, and include mRNA, cRNA,synthetic RNA and DNA, and cDNA. The polynucleotides may bedouble-stranded or single-stranded, and if single-stranded, can be thecoding strand or the non-coding (anti-sense, complementary) strand. Thepolynucleotides of the invention include the coding sequence of apolypeptide of the invention (i) in isolation, (ii) in combination withone or more additional coding sequences, so as to encode, e.g., a fusionprotein, a pre-protein, a prepro-protein, or the like, (iii) incombination with non-coding sequences, such as introns, controlelements, such as a promoter (e.g., naturally occurring or recombinantor shuffled promoter), a terminator element, or 5′ and/or 3′untranslated regions effective for expression of the coding sequence ina suitable host, and/or (iv) in a vector, cell, or host environment inwhich the coding sequence is a heterologous gene.

Polynucleotides of the invention can also be found in combination withtypical compositional formulations of nucleic acids, including in thepresence of carriers, buffers, adjuvants, excipients, and the like, asare known to those of ordinary skill in the art. Polynucleotidefragments typically comprise at least about 200 nucleotide bases, suchas at least about 250, 300, 350, 400, 450, 460, 470, or more bases. Thenucleotide fragments of polynucleotides of the invention may hybridizeunder highly stringent conditions to a polynucleotide sequence describedherein and/or encode amino acid sequences having at least one of theproperties of polypeptides of the invention described herein.

Modified Coding Sequences

As will be understood by those of ordinary skill in the art, it can beadvantageous to modify a coding sequence to enhance its expression in aparticular host. The genetic code is redundant with 64 possible codons,but most organisms preferentially use a subset of these codons. Thecodons that are utilized most often in a species are considered optimalcodons, and those not utilized very often are classified as rare orlow-usage codons (see, e.g., Zhang, S. P. et al. (1991) Gene 105:61-72).Codons can be substituted to reflect the preferred codon usage of thehost, a process sometimes termed “codon optimization” or “controllingfor species codon bias.”

Modified coding sequence containing codons preferred by a particularprokaryotic or eukaryotic host (see, e.g., Murray, E. et al. (1989) NucAcids Res 17:477-508; Griswold et al., (2003) Protein Expr. Purif.27(1):134-42) can be prepared, for example, to increase the rate oftranslation or to produce recombinant RNA transcripts having desirableproperties, such as a longer half-life, as compared with transcriptsproduced from a non-optimized sequence. Translation stop codons can alsobe modified to reflect host preference. For example, preferred stopcodons for S. cerevisiae and mammals are UAA and UGA respectively. Thepreferred stop codon for monocotyledonous plants is UGA, whereas insectsand E. coli prefer to use UAA as the stop codon (Dalphin, M. E. et al.(1996) Nucl. Acids Res. 24:216-218).

The polynucleotide sequences of the present invention can be engineeredin order to alter a coding sequence of the invention for a variety ofreasons, including but not limited to, alterations which modify thecloning, processing and/or expression of the gene product. For example,alterations may be introduced using techniques which are well known inthe art, e.g., site-directed mutagenesis, to insert new restrictionsites, to alter glycosylation patterns, to introduce or removeattachment groups (e.g., for pegylation or other conjugation), to changecodon preference, to introduce splice sites, etc.

Silent Variations

Because of the degeneracy of the genetic code, a large number offunctionally identical nucleic acids encode any given polypeptide. Forinstance, inspection of the codon table below (Table 5) shows thatcodons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acidarginine. Thus, at every position in a nucleic acid sequence where anarginine is specified by a codon, the codon can be altered to any of thecorresponding codons described above without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations”. It isto be understood that U in an RNA sequence corresponds to T in a DNAsequence. TABLE 5 Codon Table Amino acid Codon(s) Alanine Ala A GCA GCCGCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acidGlu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

It will thus be appreciated by those skilled in the art that due to thedegeneracy of the genetic code, a multitude of nucleic acids sequencesencoding polypeptides of the invention may be produced, some of whichmay bear minimal sequence identity to the nucleic acid sequencesexplicitly disclosed herein. One of ordinary skill in the art willrecognize that each codon in a nucleic acid (except AUG and UGC, whichare ordinarily the only codon for methionine and tryptophan,respectively) can be modified by standard techniques to encode afunctionally identical polypeptide. Accordingly, each silent variationof a nucleic acid which encodes a polypeptide is implicit in anydescribed sequence. The invention also provides each and every possiblevariation of a nucleic acid sequence encoding a polypeptide of theinvention that can be made by selecting combinations based on possiblecodon choices. These combinations are made in accordance with thestandard triplet (codon) genetic code (e.g., as set forth in Table 5),as applied to the nucleic acid sequence encoding a polypeptide of theinvention. All such variations of every nucleic acid herein arespecifically provided and described by consideration of the sequence incombination with the genetic code. One of skill is fully able togenerate any silent substitution of the sequences listed herein.

Using Polynucleotides

The polynucleotides of the invention have a variety of uses in, forexample, recombinant production (i.e., expression) of the polypeptidesof the invention typically through expression of a plasmid expressionvector comprising a sequence encoding the polypeptide or fragmentthereof; as therapeutics; as prophylactics; as diagnostic tools; asimmunogens; as adjuvants; as diagnostic probes for the presence ofcomplementary or partially complementary nucleic acids (including fordetection of a wild-type interferon-alpha nucleic acid), as substratesfor further reactions, e.g., recursive sequence recombination reactionsor mutation reactions to produce new and/or improved variants, and thelike.

Vectors, Promoters, and Expression Systems

The present invention also includes recombinant constructs comprisingone or more of the nucleic acid sequences as broadly described above.The constructs comprise a vector, such as, a plasmid, a cosmid, a phage,a virus, a bacterial artificial chromosome (BAC), a yeast artificialchromosome (YAC), and the like, into which a nucleic acid sequence ofthe invention has been inserted, in a forward or reverse orientation. Insome instances, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the nucleic acidsequence. Large numbers of suitable vectors and promoters are known tothose of skill in the art, and are commercially available.

General texts that describe molecular biological techniques usefulherein, including the use of vectors, promoters and many other relevanttopics, include Berger, supra; Sambrook (1989), supra, and Ausubel,supra. Examples of techniques sufficient to direct persons of skillthrough in vitro amplification methods, including the polymerase chainreaction (PCR) the ligase chain reaction (LCR), Qβ-replicaseamplification and other RNA polymerase mediated techniques (e.g.,NASBA), e.g., for the production of the homologous nucleic acids of theinvention are found in Berger, Sambrook, and Ausubel, all supra, as wellas Mullis et al. (1987) U.S. Pat. No. 4,683,202; PCR Protocols: A Guideto Methods and Applications (Innis et al., eds.) Academic Press Inc. SanDiego, Calif. (1990) (“Innis”); Arnheim & Levinson (Oct. 1, 1990) C&EN36-47; The Journal Of NIH Research (1991) 3:81-94; (Kwoh et al. (1989)Proc Natl Acad Sci USA 86:1173-1177; Guatelli et al. (1990) Proc NatlAcad Sci USA 87:1874-1878; Lomeli et al. (1989) J Clin Chem35:1826-1831; Landegren et al. (1988) Science 241:1077-1080; Van Brunt(1990) Biotechnology 8:291-294; Wu and Wallace (1989) Gene 4:560-569;Barringer et al. (1990) Gene 89:117-122, and Sooknanan and Malek (1995)Biotechnology 13:563-564. Improved methods of cloning in vitro amplifiednucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039.Improved methods of amplifying large nucleic acids by PCR are summarizedin Cheng et al. (1994) Nature 369:684-685 and the references therein, inwhich PCR amplicons of up to 40 kilobases (kb) are generated. One ofskill will appreciate that essentially any RNA can be converted into adouble stranded DNA suitable for restriction digestion, PCR expansionand sequencing using reverse transcriptase and a polymerase. SeeAusubel, Sambrook and Berger, all supra.

The present invention also provides host cells that are transduced withvectors of the invention, and the production of polypeptides of theinvention by recombinant techniques. Host cells are geneticallyengineered (e.g., transduced, transformed or transfected) with thevectors of this invention, which may be, for example, a cloning vectoror an expression vector. The vector may be, for example, in the form ofa plasmid, a viral particle, a phage, etc. The engineered host cells canbe cultured in conventional nutrient media modified as appropriate foractivating promoters, selecting transformants, or amplifying genes. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to those skilled in the art and in the references cited herein,including, e.g., Freshney (1994) Culture of Animal Cells, a Manual ofBasic Technique, third edition, Wiley-Liss, New York and the referencescited therein.

The polypeptides of the invention can also be produced in non-animalcells such as plants, yeast, fungi, bacteria and the like. In additionto Sambrook, Berger and Ausubel, details regarding cell culture arefound in, e.g., Payne et al. (1992) Plant Cell and Tissue Culture inLiquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg andPhillips (eds.) (1995) Plant Cell, Tissue and Organ Culture; FundamentalMethods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg NY);Atlas & Parks (eds.) The Handbook of Microbiological Media (1993) CRCPress, Boca Raton, Fl.

The polynucleotides of the present invention and fragments thereof maybe included in any one of a variety of expression vectors for expressinga polypeptide. Such vectors include chromosomal, nonchromosomal andsynthetic DNA sequences, e.g., derivatives of SV40, bacterial plasmids,phage DNA, baculovirus, yeast plasmids, vectors derived fromcombinations of plasmids and phage DNA, viral DNA such as vaccinia,adenovirus, fowl pox virus, pseudorabies, adeno-associated virus,retroviruses and many others. Any vector that transduces geneticmaterial into a cell, and, if replication is desired, which isreplicable and viable in the relevant host can be used.

The nucleic acid sequence in the expression vector is operatively linkedto an appropriate transcription control sequence (promoter) to directmRNA synthesis. Examples of such promoters include: LTR or SV40promoter, E. coli lac or trp promoter, phage lambda P_(L) promoter, CMVpromoter, and other promoters known to control expression of genes inprokaryotic or eukaryotic cells or their viruses. The expression vectoralso contains a ribosome binding site for translation initiation, and atranscription terminator. The vector optionally includes appropriatesequences for amplifying expression, e.g., an enhancer. In addition, theexpression vectors optionally comprise one or more selectable markergenes to provide a phenotypic trait for selection of transformed hostcells, such as dihydrofolate reductase or neomycin resistance foreukaryotic cell culture, or such as tetracycline or ampicillinresistance in E. coli.

The vector containing the appropriate DNA sequence encoding apolypeptide of the invention, as well as an appropriate promoter orcontrol sequence, may be employed to transform an appropriate host topermit the host to express the polypeptide. Examples of appropriateexpression hosts include: bacterial cells, such as E. coli,Streptomyces, and Salmonella typhimurium; fungal cells, such asSaccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa; insectcells such as Drosophila and Spodoptera frugiperda; mammalian cells suchas CHO, COS, BHK, HEK 293 or Bowes melanoma; plant cells, etc. It isunderstood that not all cells or cell lines need to be capable ofproducing fully functional polypeptides of the invention or fragmentsthereof; for example, antigenic fragments of the polypeptide may beproduced in a bacterial or other expression system. The invention is notlimited by the host cells employed.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the polypeptide or fragment thereof.For example, when large quantities of a polypeptide or fragments thereofare needed for the induction of antibodies, vectors which direct highlevel expression of fusion proteins that are readily purified may bedesirable. Such vectors include, but are not limited to, multifunctionalE. coli cloning and expression vectors such as BLUESCRIPT (Stratagene),in which the nucleotide coding sequence may be ligated into the vectorin-frame with sequences for the amino-terminal Met and the subsequent 7residues of beta-galactosidase so that a hybrid protein is produced; pINvectors (Van Heeke & Schuster (1989) J Biol Chem 264:5503-5509); pETvectors (Novagen, Madison Wis.); and the like.

Similarly, in the yeast Saccharomyces cerevisiae a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase and PGH may be used for production of the polypeptidesof the invention. For reviews, see Ausubel, supra, Berger, supra, andGrant et al. (1987) Methods in Enzymology 153:516-544.

In mammalian host cells, a number of expression systems, such asviral-based systems, may be utilized. In cases where an adenovirus isused as an expression vector, a coding sequence is optionally ligatedinto an adenovirus transcription/translation complex consisting of thelate promoter and tripartite leader sequence. Insertion in anonessential E1 or E3 region of the viral genome results in a viablevirus capable of expressing a polypeptide of the invention in infectedhost cells (Logan and Shenk (1984) Proc Natl Acad Sci USA 81:3655-3659).In addition, transcription enhancers, such as the rous sarcoma virus(RSV) enhancer, are used to increase expression in mammalian host cells.Host cells, media, expression systems, and methods of production includethose known for cloning and expression of various mammalianinterferon-alphas (e.g., human interferon-alphas).

Additional Expression Elements

Specific initiation signals can aid in efficient translation of apolynucleotide coding sequence of the invention and/or fragmentsthereof. These signals can include, e.g., the ATG initiation codon andadjacent sequences. In cases where an coding sequence, its initiationcodon and upstream sequences are inserted into the appropriateexpression vector, no additional translational control signals may beneeded. However, in cases where only coding sequence (e.g., a matureprotein coding sequence), or a portion thereof, is inserted, exogenousnucleic acid transcriptional control signals including the ATGinitiation codon must be provided. Furthermore, the initiation codonmust be in the correct reading frame to ensure transcription of theentire insert. Exogenous transcriptional elements and initiation codonscan be of various origins, both natural and synthetic. The efficiency ofexpression can enhanced by the inclusion of enhancers appropriate to thecell system in use (see, e.g., Scharf D. et al. (1994) Results ProblCell Differ 20:125-62; and Bittner et al. (1987) Methods in Enzymol153:516-544).

Secretion/Localization Sequences

Polynucleotides encoding polypeptides of the invention can also befused, for example, in-frame to nucleic acid encoding asecretion/localization sequence, to target polypeptide expression to adesired cellular compartment, membrane, or organelle, or to directpolypeptide secretion to the periplasmic space or into the cell culturemedia. Such sequences are known to those of skill, and include secretionleader or signal peptides, organelle targeting sequences (e.g., nuclearlocalization sequences, ER retention signals, mitochondrial transitsequences, chloroplast transit sequences), membrane localization/anchorsequences (e.g., stop transfer sequences, GPI anchor sequences), and thelike.

Expression Hosts

In a further aspect, the present invention relates to host cellscontaining any of the above-described nucleic acids, vectors, or otherconstructs of the invention. The host cell can be a eukaryotic cell,such as a mammalian cell, a yeast cell, or a plant cell, or the hostcell can be a prokaryotic cell, such as a bacterial cell. Introductionof the construct into the host cell can be effected by calcium phosphatetransfection, DEAE-Dextran mediated transfection, electroporation, geneor vaccine gun, injection, or other common techniques (see, e.g., Davis,L., Dibner, M., and Battey, I. (1986) Basic Methods in MolecularBiology) for in vivo, ex vivo or in vitro methods.

A host cell strain is optionally chosen for its ability to modulate theexpression of the inserted sequences or to process the expressed proteinin the desired fashion. Such modifications of the protein include, butare not limited to, acetylation, carboxylation, glycosylation,phosphorylation, lipidation and acylation. Post-translational processingwhich cleaves a “pre” or a “prepro” form of the protein may also beimportant for correct insertion, folding and/or function. Different hostcells such as E. coli, Bacillus sp., yeast or mammalian cells such asCHO, HeLa, BHK, MDCK, HEK 293, WI38, etc. have specific cellularmachinery and characteristic mechanisms for such post-translationalactivities and may be chosen to ensure the correct modification andprocessing of the introduced foreign protein.

Stable expression can be used for long-term, high-yield production ofrecombinant proteins. For example, cell lines which stably express apolypeptide of the invention are transduced using expression vectorswhich contain viral origins of replication or endogenous expressionelements and a selectable marker gene. Following the introduction of thevector, cells may be allowed to grow for 1-2 days in an enriched mediabefore they are switched to selective media. The purpose of theselectable marker is to confer resistance to selection, and its presenceallows growth and recovery of cells which successfully express theintroduced sequences. For example, resistant clumps of stablytransformed cells can be proliferated using tissue culture techniquesappropriate to the cell type.

Host cells transformed with a nucleotide sequence encoding a polypeptideof the invention are optionally cultured under conditions suitable forthe expression and recovery of the encoded protein from cell culture.The polypeptide produced by a recombinant cell may be secreted,membrane-bound, or contained intracellularly, depending on the sequenceand/or the vector used. As will be understood by those of skill in theart, expression vectors containing polynucleotides encoding polypeptidesof the invention can be designed with signal sequences which directsecretion of the mature polypeptides through a prokaryotic or eukaryoticcell membrane.

Additional Sequences

The polynucleotides of the present invention optionally comprise acoding sequence fused in-frame to a marker sequence which, e.g.,facilitates purification and/or detection of the encoded polypeptide.Such purification subsequences include, but are not limited to, metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, a sequence which binds glutathione(e.g., GST), a hemagglutinin (HA) tag (corresponding to an epitopederived from the influenza hemagglutinin protein; Wilson, I. et al.(1984) Cell 37:767), maltose binding protein sequences, the FLAG epitopeutilized in the FLAGS extension/affinity purification system, and thelike. The inclusion of a protease-cleavable polypeptide linker sequencebetween the purification domain and the polypeptide sequence is usefulto facilitate purification.

For example, one expression vector possible to use in the compositionsand methods described herein provides for expression of a fusion proteincomprising a polypeptide of the invention fused to a polyhistidineregion separated by an enterokinase cleavage site. The histidineresidues facilitate purification on IMIAC (immobilized metal ionaffinity chromatography, as described in Porath et al. (1992) ProteinExpression and Purification 3:263-281) while the enterokinase cleavagesite provides a method for separating the desired polypeptide from thepolyhistidine region. pGEX vectors (Promega; Madison, Wis.) areoptionally used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toligand-agarose beads (e.g., glutathione-agarose in the case ofGST-fusions) followed by elution in the presence of free ligand.

An additional construction in the compositions and methods describedherein provides for proteins, and their encoding nucleic acids,comprising polypeptides of the invention (or one or more fragmentsthereof), e.g., as described herein, fused to an Ig molecule, e.g.,human IgG Fc (“fragment crystallizable,” or fragment complement binding)hinge, CH2 domain and CH3 domain (and nucleotide sequences encodingthem). Fc is the portion of the antibody responsible for binding toantibody receptors on cells and the C1q component of complement. Thesefusion proteins or fragments thereof and their encoding nucleic acidsare optionally useful as prophylactic and/or therapeutic drugs or asdiagnostic tools (see also, e.g., Challita-Eid, P. et al. (1998) JImmunol 160:3419-3426; Sturmhoefel, K. et al. (1999) Cancer Res59:4964-4972).

Polypeptide Production and Recovery

Following transduction of a suitable host strain and growth of the hoststrain to an appropriate cell density, the selected promoter is inducedby appropriate means (e.g., temperature shift or chemical induction) andcells are cultured for an additional period. Cells are typicallyharvested by centrifugation, disrupted by physical or chemical means,and the resulting crude extract retained for further purification.Eukaryotic or microbial cells employed in expression of the proteins canbe disrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, orother methods, which are well know to those skilled in the art.

As noted, many references are available for the culture and productionof many cells, including cells of bacterial, plant, animal (especiallymammalian) and archebacterial origin. See, e.g., Sambrook, Ausubel, andBerger (all supra), as well as Freshney (1994) Culture of Animal Cells,a Manual of Basic Technique, third edition, Wiley-Liss, New York and thereferences cited therein; Doyle and Griffiths (1997) Mammalian CellCulture: Essential Techniques John Wiley and Sons, NY; Humason (1979)Animal Tissue Techniques, fourth edition W.H. Freeman and Company; andRicciardelli et al. (1989) In vitro Cell Dev Biol 25:1016-1024. Forplant cell culture and regeneration see, e.g., Payne et al. (1992) PlantCell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. NewYork, N.Y.; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue andOrgan Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag(Berlin Heidelberg New York) and Plant Molecular Biology (1993) R. R. D.Croy (ed.) Bios Scientific Publishers, Oxford, U.K. ISBN 0 12 198370 6.Cell culture media in general are set forth in Atlas and Parks (eds.)The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.Additional information for cell culture is found in available commercialliterature such as the Life Science Research Cell Culture Catalogue fromSigma-Aldrich, Inc (St Louis, Mo.) (“Sigma-LSRCCC”) and, e.g., the PlantCulture Catalogue and supplement also from Sigma-Aldrich, Inc (St Louis,Mo.) (“Sigma-PCCS”).

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by any of a number of methods well known inthe art, including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography (e.g., using any of the tagging systems noted herein),hydroxylapatite chromatography, and lectin chromatography. Proteinrefolding steps can be used, as desired, in completing configuration ofthe mature protein or fragments thereof. Finally, high performanceliquid chromatography (HPLC) can be employed in the final purificationsteps. In addition to the references noted, supra, a variety ofpurification methods are well known in the art, including, e.g., thoseset forth in Sandana (1997) Bioseparation of Proteins, Academic Press,Inc.; Bollag et al. (1996) Protein Methods, 2^(nd) Edition Wiley-Liss,NY; Walker (1996) The Protein Protocols Handbook Humana Press, NJ;Harris and Angal (1990) Protein Purification Applications: A PracticalApproach IRL Press at Oxford, Oxford, England; Harris and Angal ProteinPurification Methods: A Practical Approach IRL Press at Oxford, Oxford,England; Scopes (1993) Protein Purification: Principles and Practice3^(rd) Edition Springer Verlag, NY; Janson and Ryden (1998) ProteinPurification: Principles, High Resolution Methods and Applications,Second Edition Wiley-VCH, NY; and Walker (1998) Protein Protocols onCD-ROM Humana Press, NJ.

In Vitro Expression Systems

Cell-free transcription/translation systems can also be employed toproduce polypeptides of the invention using polynucleotides of thepresent invention. Several such systems are commercially available. Ageneral guide to in vitro transcription and translation protocols isfound in Tymms (1995) In vitro Transcription and Translation Protocols:Methods in Molecular Biology Volume 37, Garland Publishing, NY.

In Vivo Uses and Applications

Polynucleotides that encode a polypeptide of the invention, orcomplements of the polynucleotides (including e.g., antisense orribozyme molecules), are optionally administered to a cell to accomplisha therapeutically useful process or to express a therapeutically usefulproduct. These in vivo applications, including gene therapy, include amultitude of techniques by which gene expression may be altered incells. Such methods include, for instance, the introduction of genes forexpression of, e.g., therapeutically and/or prophylactically usefulpolypeptides, such as the polypeptides of the present invention.

In Vivo Polypeptide Expression

Polynucleotides encoding polypeptides of the invention are particularlyuseful for in vivo therapeutic applications, using techniques well knownto those skilled in the art. For example, cultured cells are engineeredex vivo with at least one polynucleotide (DNA or RNA) of the inventionand/or other polynucleotide sequences encoding, e.g., at least one of anantigen, cytokine, other co-stimulatory molecule, adjuvant, etc., andthe like, with the engineered cells then being returned to the patient.Cells may also be engineered in vivo for expression of one or morepolypeptides in vivo, including polypeptides and/or antigenic peptidesof the invention.

A number of viral vectors suitable for organismal in vivo transductionand expression are known. Such vectors include retroviral vectors (see,e.g., Miller, Curr Top Microbiol Immunol (1992) 158:1-24; Salmons andGunzburg (1993) Human Gene Therapy 4:129-141; Miller et al. (1994)Methods in Enzymology 217:581-599) and adeno-associated vectors(reviewed in Carter (1992) Curr Opinion Biotech 3:533-539; Muzcyzka(1992) Curr Top Microbiol Immunol. 158:97-129). Other viral vectors thatare used include adenoviral vectors, herpes viral vectors and Sindbisviral vectors, as generally described in, e.g., Jolly (1994) Cancer GeneTherapy 1:51-64; Latchman (1994) Molec Biotechnol 2:179-195; andJohanning et al. (1995) Nucl Acids Res 23:1495-1501.

In one aspect, a pox virus vector can be used. The pox viral vector istransfected with a polynucleotide sequence encoding a polypeptide of theinvention, such as an eIL-2 polypeptide, and is useful in prophylactic,therapeutic and diagnostic applications where enhancement of an immuneresponse, such as e.g., increased or improved T cell proliferation isdesired. See viral vectors discussed in, e.g., Berencsi et al., J InfectDis (2001) 183(8):1171-9; Rosenwirth et al., Vaccine 2001 Feb. 8;19(13-14):1661-70; Kittlesen et al., J Immunol (2000) 164(8):4204-11;Brown et al. Gene Ther 2000 7(19):1680-9; Kanesa-thasan et al., Vaccine(2000) 19(4-5):483-91; Sten (2000) Drug 60(2):249-71. Compositionscomprising such vectors and an acceptable excipient are also a featureof the invention.

Gene therapy and genetic vaccines provide methods for combating chronicinfectious diseases (e.g., HIV infection, viral hepatitis), as well asnon-infectious diseases including cancer and some forms of congenitaldefects such as enzyme deficiencies, and such methods can be employedwith polynucleotides of the invention, including, e.g., vectors andcells comprising such polynucleotides. Several approaches forintroducing nucleic acids and vectors into cells in vivo, ex vivo and invitro have been used and can be employed with polynucleotides of theinvention, and vectors comprising such polynucleotides. These approachesinclude liposome based gene delivery (Debs and Zhu (1993) WO 93/24640and U.S. Pat. No. 5,641,662; Mannino and Gould-Fogerite (1988)BioTechniques 6(7):682-691; Rose, U.S. Pat. No. 5,279,833; Brigham(1991) WO 91/06309; and Felgner et al. (1987) Proc Natl Acad Sci USA84:7413-7414; Brigham et al. (1989) Am J Med Sci 298:278-281; Nabel etal. (1990) Science 249:1285-1288; Hazinski et al. (1991) Am J Resp CellMolec Biol 4:206-209; and Wang and Huang (1987) Proc Natl Acad Sci USA84:7851-7855); adenoviral vector mediated gene delivery, e.g., to treatcancer (see, e.g., Chen et al. (1994) Proc Natl Acad Sci USA91:3054-3057; Tong et al. (1996) Gynecol Oncol 61:175-179; Clayman etal. (1995) Cancer Res. 5:1-6; O'Malley et al. (1995) Cancer Res55:1080-1085; Hwang et al. (1995) Am J Respir Cell Mol Biol 13:7-16;Haddada et al. (1995) Curr Top Microbiol Immunol. 1995 (Pt. 3):297-306;Addison et al. (1995) Proc Natl Acad Sci USA 92:8522-8526; Colak et al.(1995) Brain Res 691:76-82; Crystal (1995) Science 270:404-410; Elshamiet al. (1996) Human Gene Ther 7:141-148; Vincent et al. (1996) JNeurosurg 85:648-654), and many others. Replication-defective retroviralvectors harboring therapeutic polynucleotide sequence as part of theretroviral genome have also been used, particularly with regard tosimple MuLV vectors. See, e.g., Miller et al. (1990) Mol Cell Biol10:4239 (1990); Kolberg (1992) J NIH Res 4:43, and Cometta et al. (1991)Hum Gene Ther 2:215). Nucleic acid transport coupled to ligand-specific,cation-based transport systems (Wu and Wu (1988) J Biol Chem,263:14621-14624) has also been used. Naked DNA expression vectors havealso been described (Nabel et al. (1990), supra); Wolff et al. (1990)Science, 247:1465-1468). In general, these approaches can be adapted tothe invention by incorporating nucleic acids encoding the polypeptidesof the invention into the appropriate vectors.

General texts which describe gene therapy protocols, which can beadapted to the present invention by introducing the nucleic acids of theinvention into patients, include, e.g., Robbins (1996) Gene TherapyProtocols, Humana Press, NJ, and Joyner (1993) Gene Targeting: APractical Approach, IRL Press, Oxford, England.

Antisense Technology

In addition to expression of the nucleic acids of the invention as genereplacement nucleic acids, the nucleic acids are also useful for senseand anti-sense suppression of expression, e.g., to down-regulateexpression of a nucleic acid of the invention, once, or when, expressionof the nucleic acid is no-longer desired in the cell. Similarly, thenucleic acids of the invention, or subsequences or anti-sense sequencesthereof, can also be used to block expression of naturally occurringhomologous nucleic acids. A variety of sense and anti-sense technologiesare known in the art, e.g., as set forth in Lichtenstein and Nellen(1997) Antisense Technology: A Practical Approach IRL Press at OxfordUniversity, Oxford, England, and in Agrawal (1996) AntisenseTherapeutics Humana Press, NJ, and the references cited therein.

Use as Probes

Also contemplated are uses of polynucleotides, also referred to hereinas oligonucleotides, typically having at least 12 bases, preferably atleast 15, more preferably at least 20, at least 30, or at least 50 ormore bases, which hybridize under highly stringent conditions to apolynucleotide of the invention, or fragments thereof. Thepolynucleotides may be used as probes, primers, sense and antisenseagents, and the like, according to methods as noted supra.

Nucleic Acid Hybridization

Nucleic acids “hybridize” when they associate, typically in solution.Nucleic acids hybridize due to a variety of well characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, part I, chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays,” (Elsevier, New York)(hereinafter “Tjissen”), as well as in Ausubel, supra, Hames and Higgins(1995) Gene Probes 1, IRL Press at Oxford University Press, Oxford,England (Hames and Higgins 1) and Hames and Higgins (1995) Gene Probes2, IRL Press at Oxford University Press, Oxford, England (Hames andHiggins 2) provide details on the synthesis, labeling, detection andquantification of DNA and RNA, including oligonucleotides.

An indication that two nucleic acid sequences are substantiallyidentical is that the two molecules hybridize to each other under atleast stringent conditions. The phrase “hybridizing specifically to,”refers to the binding, duplexing, or hybridizing of a molecule only to aparticular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetpolynucleotide sequence.

“Stringent hybridization wash conditions” and “stringent hybridizationconditions” in the context of nucleic acid hybridization experiments,such as Southern and northern hybridizations, are sequence dependent,and are different under different environmental parameters. An extensiveguide to hybridization of nucleic acids is found in Tijssen (1993),supra, and in Hames and Higgins 1 and Hames and Higgins 2, supra.

For purposes of the present invention, generally, “highly stringent”hybridization and wash conditions are selected to be about 5° C. or lesslower than the thermal melting point (T_(m)) for the specific sequenceat a defined ionic strength and pH (as noted below, highly stringentconditions can also be referred to in comparative terms). The T_(m) isthe temperature (under defined ionic strength and pH) at which 50% ofthe test sequence hybridizes to a perfectly matched probe. In otherwords, the T_(m) indicates the temperature at which the nucleic acidduplex is 50% denatured under the given conditions and its represents adirect measure of the stability of the nucleic acid hybrid. Thus, theT_(m) corresponds to the temperature corresponding to the midpoint intransition from helix to random coil; it depends on length, nucleotidecomposition, and ionic strength for long stretches of nucleotides.Typically, under “stringent conditions,” a probe will hybridize to itstarget subsequence, but to no other sequences. “Very stringentconditions” are selected to be equal to the T_(m) for a particularprobe.

After hybridization, unhybridized nucleic acid material can be removedby a series of washes, the stringency of which can be adjusted dependingupon the desired results. Low stringency washing conditions (e.g., usinghigher salt and lower temperature) increase sensitivity, but can productnonspecific hybridization signals and high background signals. Higherstringency conditions (e.g., using lower salt and higher temperaturethat is closer to the hybridization temperature) lowers the backgroundsignal, typically with only the specific signal remaining. See, Rapley,R. and Walker, J. M. eds., Molecular Biomethods Handbook (Humana Press,Inc. 1998) (hereinafter “Rapley and Walker”), which is incorporatedherein by reference in its entirety for all purposes.

The T_(m) of a DNA-DNA duplex can be estimated using equation (1):T_(m) (° C.)=81.5° C.+16.6 (log₁₀ M)+0.41 (% G+C)−0.72 (% f)−500/n,where M is the molarity of the monovalent cations (usually Na+), (% G+C)is the percentage of guanosine (G) and cystosine (C ) nucleotides, (% f)is the percentage of formalize and n is the number of nucleotide bases(i.e., length) of the hybrid. See, Rapley and Walker, supra.

The T_(m) of an RNA-DNA duplex can be estimated using equation (2):T_(m) (° C.)=79.8° C.+18.5 (log₁₀ M)+0.58 (% G+C)−11.8 (% G+C)²−0.56 (%f)−820/n,where M is the molarity of the monovalent cations (usually Na+), (% G+C)is the percentage of guanosine (G) and cystosine (C) nucleotides, (% f)is the percentage of formamide and n is the number of nucleotide bases(i.e., length) of the hybrid. Id. Equations 1 and 2 above are typicallyaccurate only for hybrid duplexes longer than about 100-200 nucleotides.Id.

The Tm of nucleic acid sequences shorter than 50 nucleotides can becalculated as follows:T_(m) (° C.)=4(G+C)+2(A+T),where A (adenine), C, T (thymine), and G are the numbers of thecorresponding nucleotides.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formalin (orformamide) with 1 mg of heparin at 42° C., with the hybridization beingcarried out overnight. An example of stringent wash conditions is a0.2×SSC wash at 65° C. for 15 minutes (see Sambrook, supra, for adescription of SSC buffer). Often, the high stringency wash is precededby a low stringency wash to remove background probe signal. An examplelow stringency wash is 2×SSC at 40° C. for 15 minutes. An example ofhighly stringent wash conditions is 0.15M NaCl at 72° C. for about 15minutes. An example medium stringency wash for a duplex of, e.g., morethan 100 nucleotides, is 1×SSC at 45° C. for 15 minutes. An example lowstringency wash for a duplex of, e.g., more than 100 nucleotides, is4-6×SSC at 40° C. for 15 minutes. For short probes (e.g., about 10 to 50nucleotides), stringent conditions typically involve salt concentrationsof less than about 1.0 M Na⁺ ion, typically about 0.01 to 1.0 M Na⁺ ionconcentration (or other salts) at pH 7.0 to 8.3, and the temperature istypically at least about 30° C. Stringent conditions can also beachieved with the addition of destabilizing agents such as formamide.

In general, a signal to noise ratio of 2× or 2.5×-5× (or higher) thanthat observed for an unrelated probe in the particular hybridizationassay indicates detection of a specific hybridization. Detection of atleast stringent hybridization between two sequences in the context ofthe present invention indicates relatively strong structural similarityor homology to, e.g., the nucleic acids of the present inventionprovided in the sequence listings herein.

As noted, “highly stringent” conditions are selected to be about 5° C.or less lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. Target sequences that areclosely related or identical to the nucleotide sequence of interest(e.g., “probe”) can be identified under highly stringency conditions.Lower stringency conditions are appropriate for sequences that are lesscomplementary. See, e.g., Rapley and Walker; Sambrook, all supra.

Comparative hybridization can be used to identify nucleic acids of theinvention, and this comparative hybridization method is a preferredmethod of distinguishing nucleic acids of the invention. Detection ofhighly stringent hybridization between two nucleotide sequences in thecontext of the present invention indicates relatively strong structuralsimilarity/homology to, e.g., the nucleic acids provided in the sequencelisting herein. Highly stringent hybridization between two nucleotidesequences demonstrates a degree of similarity or homology of structure,nucleotide base composition, arrangement or order that is greater thanthat detected by stringent hybridization conditions. In particular,detection of highly stringent hybridization in the context of thepresent invention indicates strong structural similarity or structuralhomology (e.g., nucleotide structure, base composition, arrangement ororder) to, e.g., the nucleic acids provided in the sequence listingsherein. For example, it is desirable to identify test nucleic acidswhich hybridize to the exemplar nucleic acids herein under stringentconditions.

Thus, one measure of stringent hybridization is the ability to hybridizeto one of the listed nucleic acids of the invention (e.g., nucleic acidsequences SEQ ID NOS:59-88, and complementary polynucleotide sequencesthereof) under highly stringent conditions (or very stringentconditions, or ultra-high stringency hybridization conditions, orultra-ultra high stringency hybridization conditions). Stringenthybridization (including, e.g., highly stringent, ultra-high stringency,or ultra-ultra high stringency hybridization conditions) and washconditions can easily be determined empirically for any test nucleicacid.

For example, in determining highly stringent hybridization and washconditions, the hybridization and wash conditions are graduallyincreased (e.g., by increasing temperature, decreasing saltconcentration, increasing detergent concentration and/or increasing theconcentration of organic solvents, such as formalin, in thehybridization or wash), until a selected set of criteria are met. Forexample, the hybridization and wash conditions are gradually increaseduntil a probe comprising one or more nucleic acid sequences selectedfrom SEQ ID NOS:59-88, and complementary polynucleotide sequencesthereof, binds to a perfectly matched complementary target (again, anucleic acid comprising one or more nucleic acid sequences selected fromSEQ ID NOS: 59-88, and complementary polynucleotide sequences thereof),with a signal to noise ratio that is at least 2.5×, and optionally 5× ormore as high as that observed for hybridization of the probe to anunmatched target. In this case, the unmatched target is a nucleic acidcorresponding to, e.g., a known interferon-alpha nucleic acid sequence(e.g., an interferon-alpha nucleic acid sequence present in a publicdatabase such as GenBank or GENESEQ at the time of filing of the subjectapplication).

A test nucleic acid is said to specifically hybridize to a probe nucleicacid when it hybridizes at least ½ as well to the probe as to theperfectly matched complementary target, i.e., with a signal to noiseratio at least ½ as high as hybridization of the probe to the targetunder conditions in which the perfectly matched probe binds to theperfectly matched complementary target with a signal to noise ratio thatis at least about 2.5×-10×, typically 5×-10× as high as that observedfor hybridization to any of the unmatched target nucleic acids such as,e.g., a known interferon-alpha nucleic acid sequence as set forth above.For some such nucleic acids, the stringent conditions are selected suchthat a perfectly complementary oligonucleotide to the codingoligonucleotide hybridizes to the coding oligonucleotide with at leastabout a 5× higher signal to noise ratio than for hybridization of theperfectly complementary oligonucleotide to a control nucleic acidcorresponding to a known interferon-alpha sequence as set forth above.

Ultra high-stringency hybridization and wash conditions are those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of the probe to theperfectly matched complementary target nucleic acid is at least 10× ashigh as that observed for hybridization to any of the unmatched targetnucleic acids, such as, e.g., a known interferon-alpha nucleic acidsequence as set forth above. A target nucleic acid which hybridizes to aprobe under such conditions, with a signal to noise ratio of at least ½that of the perfectly matched complementary target nucleic acid is saidto bind to the probe under ultra-high stringency conditions.

Similarly, even higher levels of stringency can be determined bygradually increasing the hybridization and/or wash conditions of therelevant hybridization assay. For example, those in which the stringencyof hybridization and wash conditions are increased until the signal tonoise ratio for binding of the probe to the perfectly matchedcomplementary target nucleic acid is at least 10×, 20×, 50×, 100×, or500× or more as high as that observed for hybridization to any of theunmatched target nucleic acids, such as, e.g., a known interferon-alphanucleic acid sequence as set forth above. A target nucleic acid whichhybridizes to a probe under such conditions, with a signal to noiseratio of at least ½ that of the perfectly matched complementary targetnucleic acid is said to bind to the probe under ultra-ultra-highstringency conditions.

Target nucleic acids which hybridize to one or more nucleic acidsrepresented by SEQ ID NOS:59-88 under high, ultra-high and ultra-ultrahigh stringency conditions are a feature of the invention. Examples ofsuch nucleic acids include those with one or a few silent orconservative nucleic acid substitutions as compared to a given nucleicacid sequence.

Therapeutic Uses

Various interferon-alpha polypeptides and interferon-alpha conjugateshave been approved or are currently in clinical development fortreatment of a variety of conditions such as Chronic Hepatitis C,Chronic Hepatitis B, Hairy Cell Leukemia, Malignant Melanoma, FollicularLymphoma, Condylomata Acuminata, AIDS-related Kaposi's Sarcoma,Non-Hodgkin's Lymphoma, Chronic Melogenous Leukemia, Basal CellCarcinoma, Multiple Myeloma, carcinoid tumors, bladder cancer, Crohn'sDisease, Cutaneous T Cell Lymphoma, Renal Cell Carcinoma, MultipleSclerosis, and AIDS. Accordingly, the present invention includes amethod of treating a condition which is responsive to interferon-alpha,such as for example a condition described above, comprisingadministering to a subject afflicted with the condition a compositioncomprising a polypeptide of the invention or a conjugate of theinvention in an amount effective to ameliorate a symptom associated withthe condition. The invention also includes the use of a compositioncomprising a polypeptide of the invention or a conjugate of theinvention (i.e., a “composition of the invention”) to treat a conditionwhich is responsive to interferon-alpha, such as a condition describedabove, or any other condition which is responsive to a polypeptide ofthe invention or a conjugate of the invention.

Treatment of Viral Infections and Conditions Associated with ViralInfection

In one aspect, the invention provides a method for treating a subjectinfected with a virus, comprising administering to the subject acomposition of the invention in an amount effective to decrease thelevel of the virus in the subject and/or to ameliorate a symptom orcondition associated with the viral infection. Exemplary viralinfections contemplated for treatment methods of the invention include,but are not limited to, infection by a virus of the Flaviviridae family,such as, for example, Hepatitis C Virus, Yellow Fever Virus, West NileVirus, Japanese Encephalitis Virus, Dengue Virus, or Bovine ViralDiarrhea Virus; infection by a virus of the Hepadnaviridae family, suchas, for example, Hepatitis B Virus; infection by a virus of thePicornaviridae family, such as, for example, Encephalomyocarditis Virus,Human Rhinovirus, or Hepatitis A Virus; infection by a virus of theRetroviridae family, such as, for example, Human Immunodeficiency Virus,Simian Immunodeficiency Virus, Human T-Lymphotropic Virus, or RousSarcoma Virus; infection by a virus of the Coronaviridae family, suchas, for example, SARS coronavirus; infection by a virus of theRhabdoviridae family, such as, for example, Rabies Virus or VesicularStomatitis Virus, infection by a virus of the Paramyxoviridae family,such as, for example, Respiratory Syncytial Virus or ParainfluenzaVirus, infection by a virus of the Papillomaviridae family, such as, forexample, Human Papillomavirus, and infection by a virus of theHerpesviridae family, such as, for example, Herpes Simplex Virus.

The following provides non-limiting examples for treatment of exemplaryviral infections and diseases and conditions associated with suchinfections using polypeptides and conjugates of the invention, includingsuggested dosing schedules for polypeptides and conjugates of theinvention and approaches to monitoring the efficacy of such treatments.The dosing schedules of polypeptides or conjugates of the invention forthe treatment of other viral infections and diseases and conditionsassociated with viral infections, and approaches to monitoring theefficacy of such treatments, is ascertainable by one skilled in the art.

Hepatitis C Virus

In one aspect the invention provides a method of treating a patientinfected with Hepatitis C Virus (HCV), comprising administering to thepatient an effective amount of a composition of the invention comprisingone or more polypeptide or conjugate of the invention. The inventionalso provides a composition for use in treating a patient infected withHCV, comprising one or more polypeptide or conjugate of the inventionand a pharmaceutically acceptable carrier or excipient. A patientdiagnosed as infected with HCV includes a patient exhibiting HCV RNA inthe blood and/or exhibiting anti-HCV antibody in the serum.

A composition comprising a polypeptide of the invention will generallybe administered at a dose and frequency similar to what is employed inHCV therapeutic regimens using clinically-approved interferon-alphapolypeptides, such as, e.g. ROFERON®-A (Interferon alfa-2a, recombinant;Hoffmann-La Roche Inc.), INTRON® A (Interferon alfa-2b, recombinant;Schering Corporation), and INFERGEN® (interferon alfacon-1; InterMune,Inc.). Exemplary recommended dosing schedules of ROFERON or INTRON A forthe treatment of chronic HCV is 3 million IU (approximately 15micrograms (mcg)) three times a week by subcutaneous injection for,e.g., 24 to 48 weeks. An exemplary recommended dosing schedule ofINFERGEN for the treatment of chronic HCV is 9 mcg three times a week bysubcutaneous injection for, e.g., 24 to 48 weeks. Depending on a numberof factors (including but not limited to the activity and thepharmacokinetics of the polypeptide of the invention and the size andhealth of the patient), the polypeptide may be administered in loweramounts (such as, for example, about 2, 3, 4, 5, 6, 7, or 8 mcg) and/orless frequently (such as once per week or twice per week) than describedabove.

Likewise, a composition comprising a conjugate of the invention willgenerally be administered at a dose and frequency similar to what isemployed in HCV therapeutic regimens using clinically-approvedinterferon-alpha conjugates, such as, e.g., PEGASYS® (Peginterferonalfa-2a; Hoffmann-La Roche, Inc.) or PEG-INTRON® (peginterferon alfa-2b;Schering Corporation). An exemplary recommended dosing schedule ofPEGASYS for the treatment of chronic HCV is 180 mcg once weekly bysubcutaneous injection for, e.g., 24 to 48 weeks. Depending on a numberof factors (including but not limited to the molecular weight, activity,and pharmacokinetics of the conjugate of the invention and the size andhealth of the patient), the conjugate may be administered in loweramounts (such as, for example, about 25, 50, 75, 100, 125, or 150 mcg)and/or less frequently (such as once every 10 days, or once every 2weeks) than described above.

In some instances the polypeptide or conjugate of the invention isadministered in combination with one or more additional therapeuticagent(s). For example, the polypeptide or conjugate of the invention maybe administered in combination with a small-molecule antiviral drug suchas Ribavirin, which is sold under the names COPEGUS® (Hoffmann-La Roche,Inc) and REBETOL® (Schering Corporation). Alternatively, or in additionto a small-molecule antiviral drug, the polypeptide or conjugate of theinvention may be administered in combination with one or more additionalcytokine, such as, for example, IFN-gamma, which is sold under the nameActimmune® (interferon gamma-1b; InterMune, Inc.), IL-2, which is soldunder the name PROLEUKIN® IL-2 (aldesleukin recombinant humaninterleukin-2 (rhIL-2); Chiron Corp.), or IL-12 (interleukin-12).

The precise amount and frequency of administration of the polypeptide orconjugate of the invention will depend on a number of factors such asthe specific activity and the pharmacokinetic properties of thepolypeptide or the conjugate, as well as the nature of the conditionbeing treated (such as, the genotype of the Hepatitis C virus beingtreated), among other factors known to those of skill in the art.Normally, the dose should be capable of preventing or lessening theseverity or spread of the indication being treated. Such a dose may betermed an “effective” or “therapeutically effective” amount. It will beapparent to those of skill in the art that an effective amount of apolypeptide, conjugate or composition of the invention depends, interalia, upon the condition being treated, the dose, the administrationschedule, whether the polypeptide or conjugate or composition isadministered alone or in combination with other therapeutic agents, theserum half-life and other pharmacokinetic properties of the polypeptide,conjugate or composition, as well as the size, age, and general healthof the patient. The dosage and frequency of administration isascertainable by one skilled in the art using known techniques.

The effectiveness of treatment may be determined by measuring viralload, for example by determining the titer or level of virus in serum orplasma using methods known in the art, such as, e.g., by monitoringviral RNA levels using quantitative PCR-based tests, such as the COBASAMPLICOR® HCV Test, v2.0 or the COBAS AMPLICOR HCV MONITOR® Test, v2.0(both from Roche Diagnostics). In some instances, an effective amount ofa composition of the invention is one that is sufficient to achieve areduction in viral load by at least 2 log units, at least 3 log units,at least 4 log units, at least 5 log units, at least 6 log units or atleast 7 log units over the course of treatment, compared to the viralload prior to treatment (which is generally in the range of 10⁵-10⁷copies of HCV RNA/ml for chronic HCV patients). In some instances aneffective amount of a composition of the invention is an amount that issufficient to reduce viral load to levels which are essentiallyundetectable, such as, for example, less than about 500 copies/ml serumor less than about 100 copies/ml serum. The invention includes a methodof reducing the level of HCV RNA in serum of a patient infected withHCV, comprising administering to the patient a composition of theinvention in an amount effective to reduce the level of HCV RNA comparedto the HCV RNA level present prior to the start of treatment.

The effectiveness of treatment may alternatively or in addition bedetermined by measuring a parameter indicative of a condition associatedwith HCV infection, such as, e.g., liver damage. For example, the levelof serum alanine aminotransferase (ALT) may be measured using a standardassay. In general, an ALT level of less than about 50 internationalunits/ml (IU/ml) serum is considered normal. A higher ALT level may beindicative of ongoing liver damage. In some instances, an effectiveamount of a composition of the invention is an amount effective toreduce ALT level, in a patient with a higher than normal ALT level, toless than about 50 IU/ml of serum. Thus, the invention includes a methodof reducing the serum ALT level of a patient infected with HCVexhibiting an initial ALT level greater than 50 IU/ml, comprisingadministering to the patient a composition of the invention in an amounteffective to reduce the ALT level to less than about 50 IU/ml.

Human Immunodeficiency Virus

In another aspect the invention provides a method of treating a patientinfected with Human Immunodeficiency Virus (HIV), such as HIV-1 orHIV-2, or a disease or condition associated with HIV infection, such as,for example, AIDS-related Kaposi's sarcoma, comprising administering tothe patient an effective amount of a composition of the inventioncomprising one or more polypeptide or conjugate of the invention,optionally in association with other antiviral therapeutic agents asdescribed below. The invention also provides a composition for use intreating a patient infected with HIV or a disease or conditionassociated with HIV infection, comprising one or more polypeptide orconjugate of the invention and a pharmaceutically acceptable carrier orexcipient. A patient diagnosed as infected with HIV includes a patientexhibiting detectable levels of HIV RNA or proviral DNA in the blood,and/or exhibiting detectable levels of p24 antigen or anti-HIV antibodyin serum.

A composition comprising a polypeptide of the invention will generallybe administered at a dose and frequency similar to what is employed inHIV therapeutic regimens using interferon-alpha polypeptides such as,e.g. ROFERON®-A (Interferon alfa-2a, recombinant; Hoffmann-La RocheInc.), INTRON® A (Interferon alfa-2b, recombinant; ScheringCorporation), and INFERGEN® (interferon alfacon-1; InterMune, Inc.). Aswas noted above, exemplary recommended dosing schedules of ROFERON orINTRON A for the treatment of chronic HCV is 3 million IU (approximately15 micrograms (mcg)) three times a week by subcutaneous injection for,e.g., 24 to 48 weeks, and a exemplary recommended dosing schedule ofINFERGEN for the treatment of chronic HCV is 9 mcg three times a week bysubcutaneous injection for, e.g., 24 to 48 weeks. An exemplaryrecommended dosing schedule of ROFERON for the treatment of AIDS-relatedKaposi's sarcoma is 36 million units daily for 10 to 12 weeks, then 36million units 3 times a week. An exemplary recommended dosing scheduleof INTRON A for the treatment of AIDS-related Kaposi's sarcoma is 30million IU/m2 three times a week administered subcutaneously. Suchdosing schedules provide useful ranges for dosage of a polypeptide ofthe invention for the treatment of HIV or a disease or conditionassociated with HIV infection. Depending on a number of factors(including but not limited to the activity and the pharmacokinetics ofthe polypeptide of the invention and the size, age and health of thepatient), the polypeptide of the invention may be administered in loweramounts and/or less frequently than described above.

Likewise, a composition comprising a conjugate of the invention willgenerally be administered at a dose and frequency similar to what isemployed in HIV therapeutic regimens using interferon-alpha conjugates,such as, e.g., PEGASYS® (Peginterferon alfa-2a; Hoffmann-La Roche, Inc.)or PEG-INTRON® (peginterferon alfa-2b; Schering Corporation). Anexemplary dosing schedule of PEG-INTRON for the treatment of HIV isbetween about 1.0 mcg/kg/week and 3.0 mcg/kg/week by subcutaneousinjection for, e.g., 24 to 48 weeks. Such a dosing schedule provides auseful range for dosage of a conjugate of the invention for thetreatment of HIV. Depending on a number of factors (including but notlimited to the molecular weight, activity, and pharmacokinetics of theconjugate of the invention and the size, age and health of the patient),the conjugate may be administered in lower amounts (such as, forexample, about 0.1, 0.25, 0.50, or 0.75 mcg/kg/week) and/or lessfrequently (such as once every 10 days, or once every 2 weeks) thandescribed above.

In some instances the polypeptide or conjugate of the invention isadministered in combination with one or more additional therapeuticagent(s). Current clinical treatments of HIV-1 infection in man includemulti-drug combination therapies generally termed Highly ActiveAntiretroviral Therapy (“HAART”). The polypeptide or conjugate of theinvention may thus be administered in combination with HAART or otherantiviral therapeutic compounds. Typical components of HAART, whichinvolve various combinations of nucleoside reverse transcriptaseinhibitors (“NRTI”), non-nucleoside reverse transcriptase inhibitors(“NNRTI”) and HIV protease inhibitors (“PI”), are described, forexample, in A. M. Vandamme et al. (1998) Antiviral Chemistry &Chemotherapy, 9:187-203; “Drugs for HIV Infection” in The Medical LetterVol. 39 (Issue 1015) Dec. 5, 1997, pages 111-116; and published UnitedStates Patent Application US 20020182179 A1; each of which isincorporated by reference herein. If the HIV-infected patient is alsoinfected with HCV, the polypeptide or conjugate of the invention may beadministered in combination with an antiviral drug such as Ribavirin,which is sold under the names COPEGUS® (Hoffmann-La Roche, Inc) andREBETOL® (Schering Corporation), along with HAART.

The precise amount and frequency of administration of the polypeptide orconjugate of the invention, and administration of additional therapeuticagents such as HAART and/or Ribavirin, will depend on a number offactors such as the specific activity and the pharmacokinetic propertiesof the polypeptide or the conjugate, as well as the nature of thecondition being treated (such as, the presence of additional viralinfections such as HCV), among other factors known to those of skill inthe art. Normally, the dose should be capable of preventing or lesseningthe severity or spread of the indication being treated. Such a dose maybe termed an “effective” or “therapeutically effective” amount. It willbe apparent to those of skill in the art that an effective amount of apolypeptide, conjugate or composition of the invention depends, interalia, upon the condition being treated, the dose, the administrationschedule, whether the polypeptide or conjugate or composition isadministered alone or in combination with other therapeutic agents, theserum half-life and other pharmacokinetic properties of the polypeptide,conjugate or composition, as well as the size, age, and general healthof the patient. The dosage and frequency of administration isascertainable by one skilled in the art using known techniques.

In addition to general uses described above, a polypeptide or conjugateof the invention may be administered to the following subsets ofpatients infected with HIV: as an adjuvant therapy, for example to HAARTas described above; as monotherapy or combination therapy in early stagepatients when the viral load is generally high; as a combined anti-viraland immunodulatory agent for patients undergoing structured treatmentinterruptions (STI) or “drug holidays”; as salvage therapy in patientswhose HAART options are limited; as an antiviral method of treatment tokeep viral load in check without initiating HAART therapy in order todelay the appearance of HAART resistant virus.

The effectiveness of treatment may be determined by measuring viralload, for example by determining the titer or level of virus in serum orplasma using methods known in the art, such as, e.g., by monitoringHIV-1 viral RNA levels using quantitative RT-PCR based tests, such asthe AMPLICOR HIV-1 MONITOR® Test, v1.5 (Roche Diagnostics). In someinstances, an effective amount of a composition of the invention is onethat is sufficient to achieve a reduction in viral load by at least 0.5log units, at least 1 log unit, at least 2 log units, at least 3 logunits, at least 4 log units, at least 5 log units, at least 6 log unitsor at least 7 log units over the course of treatment, compared to theviral load prior to treatment. In some instances an effective amount ofa composition of the invention is an amount that is sufficient to reduceviral load to levels which are essentially undetectable, such as, forexample, less than about 50-100 copies HIV-1 RNA per ml serum. Theinvention includes a method of reducing the level of HIV RNA in serum ofa patient infected with HIV, comprising administering to the patient acomposition of the invention in an amount effective to reduce the levelof HIV RNA compared to the HIV RNA level present prior to the start oftreatment.

The effectiveness of treatment may alternatively or in addition bedetermined by a serum markers for HIV replication, such as the presenceof HIV p24 antigen in the blood. In some instances an effective amountof a composition of the invention is an amount that is sufficient toreduce the level of p24 antigen in the blood to 50%, 25%, 10% or 5% ofthe level present prior to the start of treatment. In some instances aneffective amount of a composition of the invention is an amount that issufficient to reduce the level of p24 antigen to a level which isessentially undetectable. The invention includes a method of reducingthe level of p24 antigen in serum of a patient infected with HIV,comprising administering to the patient a composition of the inventionin an amount effective to reduce the level of p24 antigen compared tothe p24 antigen level present prior to the start of treatment.

Hepatitis B Virus

In another aspect, the invention provides a method of treating a patientinfected with Hepatitis B Virus (HBV), comprising administering to thepatient an effective amount of a composition of the invention comprisingone or more polypeptide or conjugate of the invention. The inventionalso provides a composition for use in treating a patient infected withHBV, comprising one or more polypeptide or conjugate of the inventionand a pharmaceutically acceptable carrier or excipient.

A patient diagnosed as infected with HBV exhibits detectable hepatitis Bsurface antigen (HBsAg) in the serum. Chronic HBV infection is furthercategorized as either “replicative” or “non-replicative”. In replicativeinfection, the patient usually has a relatively high serum concentrationof viral DNA and detectable HBeAg, which is an alternatively processedprotein of the HBV pre-core gene that is synthesized under conditions ofhigh viral replication. However, in rare strains of HBV with mutationsin the pre-core gene, replicative infection can occur in the absence ofdetectable serum HBeAg. Patients with chronic hepatitis B andreplicative infection have a generally worse prognosis and a greaterchance of developing cirrhosis and/or hepatocellular carcinoma thanthose without HBeAg. In non-replicative infection, the rate of viralreplication in the liver is low, serum HBV DNA concentration isgenerally low and hepatitis Be antigen (HBeAg) is not detected.

A composition comprising a polypeptide of the invention will generallybe administered at a dose and frequency similar to what is employed inHBV therapeutic regimens using clinically-approved interferon-alphapolypeptides, such as, e.g. INTRON® A (Interferon alfa-2b, recombinant;Schering Corporation). An exemplary recommended dosing schedule ofINTRON A for the treatment of chronic HBV in adults is 30 to 35 millionIU per week by subcutaneous or intramuscular injection, either as 5million IU per day (qd) or as 10 million IU three times per week (tiw)for 16 weeks. Depending on a number of factors (including, but notlimited to, the activity and the pharmacokinetics of the polypeptide ofthe invention, and the size and health of the patient), the polypeptideof the invention may be administered in lower amounts (such as, forexample, about 5, 10, 15, 20, or 25 million IU per week) and/or lessfrequently (such as once per week or twice per week) than describedabove.

Likewise, a composition comprising a conjugate of the invention willgenerally be administered at a dose and frequency similar to what isemployed in HBV therapeutic regimens using interferon-alpha conjugatescurrently undergoing clinical trials, such as, e.g., PEGASYS®(Peginterferon alfa-2a; Hoffmann-La Roche, Inc.). Exemplary dosingschedules of PEGASYS for the treatment of chronic HBV is between 90mcg-270 mcg injected once per week for a total of 24 weeks. Depending ona number of factors (including but not limited to the molecular weight,activity, and pharmacokinetics of the conjugate of the invention and thesize and health of the patient), the conjugate may be administered inlower amounts (such as, for example, about 25, 50, 75, 100, 125, 150, or200 mcg) and/or less frequently (such as once every 10 days, or onceevery 2 weeks) than described above.

In some instances the polypeptide or conjugate of the invention isadministered in combination with one or more additional therapeuticagent(s). For example, the polypeptide or conjugate of the invention maybe administered in combination with antiviral drugs such as lamivudine(also known as 3TC), which is sold under the name Epivir-HBV®(GlaxoSmithKline), or adefovir dipivoxil, which is sold under the nameHepsera® (Gilead Sciences).

The precise amount and frequency of administration of the polypeptide orconjugate of the invention will depend on a number of factors such asthe specific activity and the pharmacokinetic properties of thepolypeptide or the conjugate, as well as the nature of the conditionbeing treated (such as, e.g., in the case of chronic HBV infection,whether the infection is replicative or non-replicative), among otherfactors known to those of skill in the art. Normally, the dose should becapable of preventing or lessening the severity or spread of theindication being treated. Such a dose may be termed an “effective” or“therapeutically effective” amount. It will be apparent to those ofskill in the art that an effective amount of a polypeptide, conjugate orcomposition of the invention depends, inter alia, upon the conditionbeing treated, the dose, the administration schedule, whether thepolypeptide or conjugate or composition is administered alone or incombination with other therapeutic agents, the serum half-life and otherpharmacokinetic properties of the polypeptide, conjugate or composition,as well as the size, age, and general health of the patient. The dosageand frequency of administration is ascertainable by one skilled in theart using known techniques.

The effectiveness of treatment may be determined for example bymeasuring the viral load, e.g. the level of viral DNA in serum orplasma, using methods known in the art. Methods for monitoring HBV DNAlevels include quantitative PCR-based tests, such as the COBAS AMPLICORHBV MONITOR® Test, v2.0 or the AMPLICOR HBV MONITOR® Test, v2.0 (bothfrom Roche Diagnostics). In some instances an effective amount of acomposition of the invention is an amount that is sufficient to reduceviral DNA to, e.g., less than about 500,000 copies/ml serum or less thanabout 100,000 copies/ml serum or less than about 10,000 copies/ml serum,or to levels which are essentially undetectable (such as, for example,less than about 1000 copies/ml serum, less than about 500 copies/mlserum, or less than about 200 copies/ml serum). The invention includes amethod of reducing the level of HBV DNA in serum of a patient infectedwith HBV, comprising administering to the patient a composition of theinvention in an amount effective to reduce the level of HBV DNA comparedto the HBV DNA level present prior to the start of treatment.

The effectiveness of treatment may alternatively or in addition bedetermined by measuring other serum markers for HBV replication, such asHBeAg. In some instances an effective amount of a composition of theinvention is an amount that is sufficient to reduce the level of HBeAgin serum to 50%, 25%, 10% or 5% of the level present prior to the startof treatment. In some instances an effective amount of a composition ofthe invention is an amount that is sufficient to reduce the level ofHBeAg to a level which is essentially undetectable. The inventionincludes a method of reducing the level of HBeAg in serum of a patientinfected with HBV, comprising administering to the patient a compositionof the invention in an amount effective to reduce the level of HBeAgcompared to the HBeAg level present prior to the start of treatment.

As discussed above, another serum marker indicative of HBV infection isHBsAg. Thus, the effectiveness of treatment may alternatively or inaddition be determined by measuring the level of HBsAg in the serum. Insome instances an effective amount of a composition of the invention isan amount that is sufficient to reduce the level of HBsAg in serum to50%, 25%, 10% or 5% of the level present prior to the start oftreatment. In some instances an effective amount of a composition of theinvention is an amount that is sufficient to reduce level of HBsAg to alevel which is essentially undetectable. The invention includes a methodof reducing the level of HBsAg in serum of a patient infected with HBV,comprising administering to the patient a composition of the inventionin an amount effective to reduce the level of HBsAg compared to theHBsAg level present prior to the start of treatment.

The effectiveness of treatment may alternatively or in addition bedetermined by measuring a parameter indicative of a condition associatedwith HBV infection, such as, e.g., liver damage. For example, the levelof serum alanine aminotransferase (ALT) may be measured using a standardassay. In general, an ALT level of less than about 50 internationalunits/ml (IU/ml) serum is considered normal. A higher ALT level may beindicative of ongoing liver damage. In some instances, an effectiveamount of a composition of the invention is an amount effective toreduce ALT level, in a patient with a higher than normal ALT level, toless than about 50 IU/ml of serum. Thus, the invention includes a methodof reducing the serum ALT level of a patient infected with HBVexhibiting an initial ALT level greater than 50 IU/ml, comprisingadministering to the patient a composition of the invention in an amounteffective to reduce the ALT level to less than about 50 IU/ml.

Human T-Lymphotropic Virus type 1

In another aspect the invention provides a method of treating a patientinfected with Human T-Lymphotropic Virus type 1 (HTLV-1), or a diseaseor condition associated with HTLV-1 infection, such as, for example,adult T-cell leukemia/lymphoma (ATLL), HTLV-1-associated myelopathy(HAM), Tropical Spastic Paraparesis (TSP), uveitis, or arthropathy. Themethod comprises administering to the patient an effective amount of acomposition of the invention comprising one or more polypeptide orconjugate of the invention. The invention also provides a compositionfor use in treating a patient infected with HTLV-1, or a disease orcondition associated with HTLV-1 infection, the composition comprisingone or more polypeptide or conjugate of the invention and apharmaceutically acceptable carrier or excipient. A patient diagnosedwith HTLV-1 infection includes a patient exhibiting HTLV-1 proviral DNAin the blood and/or antibody to an HTLV-1 antigen in the serum.

A composition comprising a polypeptide of the invention will generallybe administered at a dose and frequency similar to what is employed inHCV or oncology therapeutic regimens using clinically-approvedinterferon-alpha polypeptides, such as, e.g. ROFERON®-A (Interferonalfa-2a, recombinant; Hoffmann-La Roche Inc.) and INTRON® A (Interferonalfa-2b, recombinant; Schering Corporation). Exemplary recommendeddosing schedules of ROFERON or INTRON A for the treatment of chronic HCVis 3 million IU (approximately 15 micrograms (mcg)) three times a weekby subcutaneous injection for, e.g., 24 to 48 weeks. An exemplaryrecommended dosing schedule of ROFERON for the treatment of hairy-cellleukemia is 3-5 million units daily by subcutaneous injection for 16 to24 weeks, then 3 million units 3 times a week for maintenance. Anexemplary recommended dosing schedule of INTRON A for the treatment ofhairy-cell leukemia is 2 million IU/m2 (square meter of body surface)administered subcutaneously 3 times a week for 6 months. Such dosingschedules provide useful ranges for dosage of a polypeptide of theinvention for the treatment of HTLV-1 infection, or a disease orcondition associated with HTLV-1 infection such as adult T-cellleukemia/lymphoma (ATLL), HTLV-1-associated myelopathy (HAM), orTropical Spastic Paraparesis (TSP). Depending on a number of factors(including but not limited to the activity and the pharmacokinetics ofthe polypeptide of the invention and the size, age and health of thepatient), the polypeptide may be administered in lower amounts and/orless frequently than described above.

Likewise, a composition comprising a conjugate of the invention willgenerally be administered at a dose and frequency similar to what isemployed in HCV therapeutic or oncology therapeutic regimens usingclinically-approved interferon-alpha conjugates, such as, e.g., PEGASYS®(Peginterferon alfa-2a; Hoffmann-La Roche, Inc.) or PEG-INTRON®(peginterferon alfa-2b; Schering Corporation). An exemplary recommendeddosing schedule of PEGASYS for the treatment of chronic HCV is 180 mcgonce weekly by subcutaneous injection for, e.g., 24 to 48 weeks. Anexemplary recommended dosing schedule of PEG-INTRON for the treatment ofchronic myelogenous leukemia is 6 mcg/kg body weight once weekly bysubcutaneous injection for, e.g., 52 weeks. Such dosing schedulesprovide useful ranges for dosage of a conjugate of the invention for thetreatment of HTLV-1 infection, or a disease or condition associated withHTLV-1 infection such as adult T-cell leukemia/lymphoma (ATLL),HTLV-1-associated myelopathy (HAM), or Tropical Spastic Paraparesis(TSP). Depending on a number of factors (including but not limited tothe molecular weight, activity, and pharmacokinetics of the conjugate ofthe invention and the size, age and health of the patient), theconjugate may be administered in lower amounts and/or less frequentlythan described above.

In some instances the polypeptide or conjugate of the invention isadministered in combination with one or more additional therapeuticagent(s). For example, the polypeptide or conjugate of the invention maybe administered in combination with an antiretroviral drug such aszidovudine (AZT) and/or lamivudine (3TC). It may also be administered incombination with peripheral blood stem cell transplantation,conventional chemotherapy, or high dose chemotherapy with autologous orallogeneic bone marrow transplantation. Alternatively, the polypeptideor conjugate of the invention may be combined with other immunotherapy,for example with anti-interleukin-2 receptor monoclonal antibodies orinjection of cytotoxic T-cells directed against virus antigens.

The precise amount and frequency of administration of the polypeptide orconjugate of the invention will depend on a number of factors such asthe specific activity and the pharmacokinetic properties of thepolypeptide or the conjugate, as well as the nature of the conditionbeing treated, among other factors known to those of skill in the art.Normally, the dose should be capable of preventing or lessening theseverity or spread of the indication being treated. Such a dose may betermed an “effective” or “therapeutically effective” amount. It will beapparent to those of skill in the art that an effective amount of apolypeptide, conjugate or composition of the invention depends, interalia, upon the condition being treated, the dose, the administrationschedule, whether the polypeptide or conjugate or composition isadministered alone or in combination with other therapeutic agents, theserum half-life and other pharmacokinetic properties of the polypeptide,conjugate or composition, as well as the size, age, and general healthof the patient. The dosage and frequency of administration isascertainable by one skilled in the art using known techniques.

The effectiveness of treatment may be determined by measuring the HTLV-1viral load, such as, for example, measuring the level of HTLV-1 proviralDNA in the blood using methods known in the art, for example byquantitative PCR as described by Saito et al., (2004) J. Infect Dis.189(1):29-40. In some instances, an effective amount of a composition ofthe invention is one that is sufficient to achieve a reduction in viralload by at least 0.5 log unit, such as at least 1 log unit, at least 2log units, at least 3 log units, at least 4 log units, at least 5 logunits, at least 6 log units, or at least 7 log units over the course oftreatment, compared to the viral load prior to treatment. In someinstances an effective amount of a composition of the invention is anamount that is sufficient to reduce viral load to levels which areessentially undetectable. The invention includes a method of reducingthe level of HTLV-1 proviral DNA in blood of a patient infected withHTLV-1, comprising administering to the patient a composition of theinvention in an amount effective to reduce the level of HTLV-1 proviralDNA compared to that present prior to the start of treatment.

The effectiveness of treatment may alternatively or in addition bedetermined by measuring titer of an anti-HTLV-1 antibody in the serum,using methods known in the art, such as, for example, bycommercially-available tests such as INNO-LIA™ HTLV I/II (Innogenetics;Gent Belgium) and Abbott HTLV-I/HTLV-II EIA (Abbott Laboratories; AbbottPark, Ill.). In some instances an effective amount of a composition ofthe invention is an amount that is sufficient to reduce the titer of ananti-HTLV-1 antibody in the serum to 50%, 25%, 10% or 5% of the titerpresent prior to the start of treatment. In some instances an effectiveamount of a composition of the invention is an amount that is sufficientto reduce the titer of an anti-HTLV-1 antibody in the serum to a levelwhich is essentially undetectable. The invention includes a method ofreducing the titer of an anti-HTLV-1 antibody in the serum of a patientinfected with HTLV-1, comprising administering to the patient acomposition of the invention in an amount effective to reduce the titerof the anti-HTLV-1 antibody in the serum compared to that present priorto the start of treatment.

Human Papillomavirus

In another aspect the invention provides a method of treating a patientinfected with Human Papillomavirus (HPV), or a disease or conditionassociated with HPV infection, such as, for example, warts of the handsand feet, and lesions of the mucous membranes of the oral, anal andgenital cavities. While many types of HPV are relatively harmless, manyother types are spread through sexual contact and give rise to genitalor venereal warts (termed condylomata acuminata) which may give rise tocervical cancer and other genital cancers. The method comprisesadministering to the patient infected with HPV an effective amount of acomposition of the invention comprising one or more polypeptide orconjugate of the invention. The invention also provides a compositionfor use in treating a patient infected with HPV, or a disease orcondition associated with HPV infection, the composition comprising oneor more polypeptide or conjugate of the invention and a pharmaceuticallyacceptable carrier or excipient. A patient diagnosed with HPV infectionincludes a patient exhibiting HPV viral DNA in biopsied genital tissue,and sometimes (but not always) exhibiting visible lesions on genitaltissues.

A composition comprising a polypeptide of the invention will generallybe administered at a dose and frequency similar to what is employed inHPV therapeutic regimens using clinically-approved interferon-alphapolypeptides, such as, for example, INTRON® A (Interferon alfa-2b,recombinant; Schering Corporation). A recommended dose of INTRON A forthe treatment of condylomata acuminata is 1.0 million IU injected intoeach lesion, for up to 5 lesions, using a tuberculin or similar syringeand a 25-to 30-gauge needle, three times per week on alternate days, for3 weeks. Patients with 6 to 10 condylomata may receive a second(sequential) course of treatment at the above dosage schedule, to treatup to five additional condylomata per course of treatment. Patients withgreater than 10 condylomata may receive additional sequences dependingon how large a number of condylomata are present. The interferon mayalternatively or in addition be applied topically, e.g. in a cream orointment form (as described for example in Stentella et al. (1996) Clin.Exp. Obstet. Gynecol. 23(1):29-36). Such dosing schedules provide usefulranges for dosage of a polypeptide of the invention for the treatment ofHPV infection, or a disease or condition associated with HPV infectionsuch as condylomata acuminata. Depending on a number of factors(including but not limited to the activity and the pharmacokinetics ofthe polypeptide of the invention and the size, age and health of thepatient), the polypeptide of the invention may be administered in loweramounts and/or less frequently than described above. Likewise, acomposition comprising a conjugate of the invention will generally beadministered, e.g. intralesionally or topically, at a dose effective toreduce the amount of HPV viral DNA in the effected tissues or to reducethe size/or number of genital lesions in the infected individual.

In some instances the polypeptide or conjugate of the invention isadministered in combination with one or more additional therapeuticagent(s). For example, the polypeptide or conjugate of the invention maybe administered in combination with an anti-HPV therapeutic such asPodofilox (Condylox) and/or Podophyllin (Pododerm, Podocon-25).

The precise amount and frequency of administration of the polypeptide orconjugate of the invention will depend on a number of factors such asthe specific activity and the pharmacokinetic properties of thepolypeptide or the conjugate, as well as the nature of the conditionbeing treated, among other factors known to those of skill in the art.Normally, the dose should be capable of preventing or lessening theseverity or spread of the indication being treated. Such a dose may betermed an “effective” or “therapeutically effective” amount. It will beapparent to those of skill in the art that an effective amount of apolypeptide, conjugate or composition of the invention depends, interalia, upon the condition being treated, the dose, the administrationschedule, whether the polypeptide or conjugate or composition isadministered alone or in combination with other therapeutic agents, theserum half-life and other pharmacokinetic properties of the polypeptide,conjugate or composition, as well as the size, age, and general healthof the patient. The dosage and frequency of administration isascertainable by one skilled in the art using known techniques.

The effectiveness of treatment may be determined by measuring the HPVviral load, such as, for example, measuring the level of HPV viral DNAin biopsied tissue. In some instances, an effective amount of acomposition of the invention is one that is sufficient to achieve areduction in viral load by at least 0.5 log unit, such as at least 1 logunit, at least 2 log units, at least 3 log units, at least 4 log units,at least 5 log units, at least 6 log units, or at least 7 log units overthe course of treatment, compared to the viral load prior to treatment.In some instances an effective amount of a composition of the inventionis an amount that is sufficient to reduce viral load to levels which areessentially undetectable. The invention includes a method of reducingthe level of HPV viral DNA in tissue of a patient infected with HPV,comprising administering to the patient a composition of the inventionin an amount effective to reduce the level of HPV viral DNA compared tothat present prior to the start of treatment.

The effectiveness of treatment may alternatively or in addition bedetermined by observing the size or number of genital lesions(condylomata) in the infected individual. In some instances an effectiveamount of a composition of the invention is an amount that is sufficientto reduce the size and/or number of condylomata in the infectedindividual. The invention includes a method of reducing reduce the sizeand/or number of condylomata in a patient infected with HPV, comprisingadministering to the patient a composition of the invention in an amounteffective to reduce the size and/or number of condylomata in the patientcompared to those present prior to the start of treatment.

Formulations and Routes of Administration

Therapeutic formulations of the polypeptide or conjugate of theinvention are typically administered in a composition that includes oneor more pharmaceutically acceptable carriers or excipients. Suchpharmaceutical compositions may be prepared in a manner known per se inthe art to result in a polypeptide pharmaceutical that is sufficientlystorage-stable and is suitable for administration to humans or animals.

Drug Form

The polypeptide or conjugate of the invention can be used “as is” and/orin a salt form thereof. Suitable salts include, but are not limited to,salts with alkali metals or alkaline earth metals, such as sodium,potassium, calcium and magnesium, as well as e.g. zinc salts. Thesesalts or complexes may by present as a crystalline and/or amorphousstructure.

Excipients

“Pharmaceutically acceptable” means a carrier or excipient that at thedosages and concentrations employed does not cause any untoward effectsin the patients to whom it is administered. Such pharmaceuticallyacceptable carriers and excipients are well known in the art (seeRemington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed.,Mack Publishing Company (1990); Pharmaceutical Formulation Developmentof Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor &Francis (2000); and Handbook of Pharmaceutical Excipients, 3rd edition,A. Kibbe, Ed., Pharmaceutical Press (2000)).

Mix of Drugs

The composition of the invention may be administered alone or inconjunction with other therapeutic agents. Ribavirin, for example, isoften co-administered with IFN-alpha and has been shown to increaseefficacy in antiviral treatments, such as HCV treatment. A variety ofsmall molecules are being developed against both viral targets (viralproteases, viral polymerase, assembly of viral replication complexes)and host targets (host proteases required for viral processing, hostkinases required for phosphorylation of viral targets such as NS5A andinhibitors of host factors required to efficiently utilize the viralIRES). Other cytokines may be co-administered, such as for example IL-2,IL-12, IL-23, IL-27, or IFN-gamma. These agents may be incorporated aspart of the same pharmaceutical composition or may be administeredseparately from the polypeptide or conjugate of the invention, eitherconcurrently or in accordance with another treatment schedule. Inaddition, the polypeptide, conjugate or composition of the invention maybe used as an adjuvant to other therapies.

Patients

A “patient” for the purposes of the present invention includes bothhumans and other mammals. Thus the methods are applicable to both humantherapy and veterinary applications.

Types of Composition and Administration Route

The pharmaceutical composition comprising the polypeptide or conjugateof the invention may be formulated in a variety of forms, e.g. as aliquid, gel, lyophilized, or as a compressed solid. The preferred formwill depend upon the particular indication being treated and will beapparent to one skilled in the art.

The administration of the formulations of the present invention can beperformed in a variety of ways, including, but not limited to, orally,subcutaneously, intravenously, intracerebrally, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,vaginally, rectally, intraocularly, or in any other acceptable manner.The formulations can be administered continuously by infusion, althoughbolus injection is acceptable, using techniques well known in the art,such as pumps (e.g., subcutaneous osmotic pumps) or implantation. Insome instances the formulations may be directly applied as a solution orspray.

Parenterals

An example of a pharmaceutical composition is a solution designed forparenteral administration. Although in many cases pharmaceuticalsolution formulations are provided in liquid form, appropriate forimmediate use, such parenteral formulations may also be provided infrozen or in lyophilized form. In the former case, the composition mustbe thawed prior to use. The latter form is often used to enhance thestability of the active compound contained in the composition under awider variety of storage conditions, as it is recognized by thoseskilled in the art that lyophilized preparations are generally morestable than their liquid counterparts. Such lyophilized preparations arereconstituted prior to use by the addition of one or more suitablepharmaceutically acceptable diluents such as sterile water for injectionor sterile physiological saline solution.

Parenterals may be prepared for storage as lyophilized formulations oraqueous solutions by mixing, as appropriate, the polypeptide having thedesired degree of purity with one or more pharmaceutically acceptablecarriers, excipients or stabilizers typically employed in the art (allof which are termed “excipients”), for example buffering agents,stabilizing agents, preservatives, isotonifiers, non-ionic detergents,antioxidants and/or other miscellaneous additives.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They are typically present at a concentrationranging from about 2 mM to about 50 mM. Suitable buffering agents foruse with the present invention include both organic and inorganic acidsand salts thereof such as citrate buffers (e.g., monosodiumcitrate-disodium citrate mixture, citric acid-trisodium citrate mixture,citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additional possibilities are phosphatebuffers, histidine buffers and trimethylamine salts such as Tris.

Preservatives are added to retard microbial growth, and are typicallyadded in amounts of about 0.2%-1% (w/v). Suitable preservatives for usewith the present invention include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalkonium halides (e.g. benzalkonium chloride, bromide oriodide), hexamethonium chloride, alkyl parabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol and 3-pentanol.

Isotonicifiers are added to ensure isotonicity of liquid compositionsand include polyhydric sugar alcohols, preferably trihydric or highersugar alcohols, such as glycerin, erythritol, arabitol, xylitol,sorbitol and mannitol. Polyhydric alcohols can be present in an amountbetween 0.1% and 25% by weight, typically 1% to 5%, taking into accountthe relative amounts of the other ingredients.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, omithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur-containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglyceroland sodium thiosulfate; low molecular weight polypeptides (i.e. <10residues); proteins such as human serum albumin, bovine serum albumin,gelatin or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructoseand glucose; disaccharides such as lactose, maltose and sucrose;trisaccharides such as raffinose, and polysaccharides such as dextran.Stabilizers are typically present in the range of from 0.1 to 10,000parts by weight based on the active protein weight.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe present to help solubilize the therapeutic agent as well as toprotect the therapeutic polypeptide against agitation-inducedaggregation, which also permits the formulation to be exposed to shearsurface stress without causing denaturation of the polypeptide. Suitablenon-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers(184, 188 etc.), Pluronic® polyols, polyoxyethylene sorbitan monoethers(Tween®-20, Tween®-80, etc.).

Additional miscellaneous excipients include bulking agents or fillers(e.g. starch), chelating agents (e.g. EDTA), antioxidants (e.g.,ascorbic acid, methionine, vitamin E) and cosolvents.

The active ingredient may also be entrapped in microcapsules prepared,for example, by coascervation techniques or by interfacialpolymerization, for example hydroxymethylcellulose, gelatin orpoly-(methylmethacylate) microcapsules, in colloidal drug deliverysystems (for example liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules) or in macroemulsions. Such techniquesare disclosed in Remington's Pharmaceutical Sciences, supra.

In one aspect of the invention the composition is a liquid composition,such as an aqueous composition, and comprises a sulfoalkyl ethercyclodextrin derivative of the formula

wherein n is 4, 5 or 6; R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉ are each,independently, —O— or a —O—(C₂-C₆ alkyl)-SO₃—0 group, wherein at leastone of R₁, R₂ or R₃ is independently a —O—(C₂-C₆ alkyl)-SO₃— group; andS₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ are each, independently, apharmaceutically acceptable cation, including H⁺.

It should be noted that when n=4, the sulfoalkyl ether cyclodextrin mayalso be referred to as a α-sulfoalkyl ether cyclodextrin. In a similarway, when n=5, the term β-sulfoalkyl ether cyclodextrin may be employedand when n=6, the sulfoalkyl ether cyclodextrin may also be referred toas a γ-sulfoalkyl ether cyclodextrin.

In a further embodiment, n is 5 or 6. In a preferred embodiment n=6.

In a still further embodiment R₁, R₂ or R₃ is independently selectedfrom the group consisting of —OCH₂CH₂CH₂SO₃—, —OCH₂CH₂CH₂CH₂SO₃— and—OCH₂CH₂CH₂CH₂CH₂SO₃—. Most preferably, R₁, R₂ or R₃ is independently—OCH₂CH₂CH₂CH₂SO₃—.

In a further embodiment S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ are each,independently, a pharmaceutically acceptable cation selected from H⁺,alkali metals (e.g. Li⁺, Na⁺, K⁺), alkaline earth metals (e.g., Ca⁺²,Mg⁺²), ammonium ions and amine cations such as the cations of (C₁-C₆)alkylamines, piperidine, pyrazine, (C₁-C₆) alkanolamine and(C₄-C₈)cycloalkanolamine. Most preferably, S₁, S₂, S₃, S₄, S₅, S₆, S₇,S₈, and S₉ are each, independently, a pharmaceutically acceptable cationselected from the group consisting of H⁺, Li⁺, Na⁺, K⁺, in particularNa⁺.

The sulfoalkyl ether cyclodextrin may contain from 1 to 18 sulfoalkylgroups (when n=4), from 1-21 sulfoalkyl groups (when n=5) or from 1-21(when n=6). In a preferred embodiment of the invention n=5 and thesulfoalkylether derivative comprises, on average, 2-20 sulfoalkyl groups(in particular sulfobutyl groups), such as 3-10 sulfoalkyl groups (inparticular sulfobutyl groups), more preferably 4-9 sulfoalkyl groups (inparticular sulfobutyl groups), even more preferably 5-9 sulfoalkylgroups (in particular sulfobutyl groups), such as 6-8 sulfoalkyl groups(in particular sulfobutyl groups), e.g. 7 sulfoalkyl groups (inparticular sulfobutyl groups).

In some instances the sulfoalkyl ether cyclodextrin derivative is asalt, in particular a sodium salt, of β-cyclodextrin sulfobutyl ether(i.e. n=5), which on average contains 7 sulfobutyl groups. Thissulfoalkyl ether cyclodextrin derivative is also termed SBE7-β-CD and isavailable as Captisol® (Cydex, Overland Park, Kans.).

The term “C₁-C₆ alkyl” represents a branched or straight alkyl grouphaving from one to six carbon atoms. Typical C₁-C₆ alkyl groups include,but are not limited to, methyl ethyl, n-propyl, iso-propyl, n-butyl,iso-butyl, sec-butyl, tert-butyl, n-pentyl, iso-pentyl, n-hexyl andiso-hexyl.

The term “C₂-C₆ alkyl” represents a branched or straight alkyl grouphaving from two to six carbon atoms. Typical C₂-C₆ alkyl groups include,but are not limited to, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,sec-butyl, tert-butyl, n-pentyl, iso-pentyl, n-hexyl and iso-hexyl.

Further details concerning compositions comprising the polypeptidesdisclosed herein and sulfoalkyl ether cyclodextrin derivatives can befound in WO 03/002152, particularly the section entitled “The sulfoalkylether cyclodextrin derivative” on pp. 37-49, incorporated herein byreference.

Parenteral formulations to be used for in vivo administration must besterile. This is readily accomplished, for example, by filtrationthrough sterile filtration membranes.

Sustained Release Preparations

Suitable examples of sustained-release preparations includesemi-permeable matrices of solid hydrophobic polymers containing thepolypeptide or conjugate, the matrices having a suitable form such as afilm or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) orpoly(vinylalcohol)), polylactides, copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the ProLease® technology orLupron Depot® (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid. While polymers such as ethylene-vinyl acetate and lacticacid-glycolic acid enable release of molecules for long periods such asup to or over 100 days, certain hydrogels release proteins for shortertime periods. When encapsulated polypeptides remain in the body for along time, they may denature or aggregate as a result of exposure tomoisture at 37° C., resulting in a loss of biological activity andpossible changes in immunogenicity. Rational strategies can be devisedfor stabilization depending on the mechanism involved. For example, ifthe aggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Oral Administration

For oral administration, the pharmaceutical composition may be in solidor liquid form, e.g. in the form of a capsule, tablet, suspension,emulsion or solution. The pharmaceutical composition is preferably madein the form of a dosage unit containing a given amount of the activeingredient. A suitable daily dose for a human or other mammal may varywidely depending on the condition of the patient and other factors, butcan be determined by persons skilled in the art using routine methods.

Solid dosage forms for oral administration may include capsules,tablets, suppositories, powders and granules. In such solid dosageforms, the active compound may be admixed with at least one inertdiluent such as sucrose, lactose, or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances, e.g. lubricatingagents such as magnesium stearate. In the case of capsules, tablets andpills, the dosage forms may also comprise buffering agents. Tablets andpills can additionally be prepared with enteric coatings.

The polypeptides or conjugates may be admixed with adjuvants such aslactose, sucrose, starch powder, cellulose esters of alkanoic acids,stearic acid, talc, magnesium stearate, magnesium oxide, sodium andcalcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodiumalginate, polyvinyl-pyrrolidine, and/or polyvinyl alcohol, and tabletedor encapsulated for conventional administration. Alternatively, they maybe dissolved in saline, water, polyethylene glycol, propylene glycol,ethanol, oils (such as corn oil, peanut oil, cottonseed oil or sesameoil), tragacanth gum, and/or various buffers. Other adjuvants and modesof administration are well known in the pharmaceutical art. The carrieror diluent may include time delay material, such as glycerylmonostearate or glyceryl distearate alone or with a wax, or othermaterials well known in the art.

The pharmaceutical compositions may be subjected to conventionalpharmaceutical operations such as sterilization and/or may containconventional adjuvants such as preservatives, stabilizers, wettingagents, emulsifiers, buffers, fillers, etc., e.g. as disclosed elsewhereherein.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants such as wetting agents,sweeteners, flavoring agents and perfuming agents.

Pulmonary Delivery

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise the polypeptide or conjugatedissolved in water at a concentration of, e.g., about 0.01 to 25 mg ofconjugate per mL of solution, preferably about 0.1 to 10 mg/mL. Theformulation may also include a buffer and a simple sugar (e.g., forprotein stabilization and regulation of osmotic pressure), and/or humanserum albumin ranging in concentration from 0.1 to 10 mg/ml. Examples ofbuffers that may be used are sodium acetate, citrate and glycine.Preferably, the buffer will have a composition and molarity suitable toadjust the solution to a pH in the range of 3 to 9. Generally, buffermolarities of from 1 mM to 50 mM are suitable for this purpose. Examplesof sugars which can be utilized are lactose, maltose, mannitol,sorbitol, trehalose, and xylose, usually in amounts ranging from 1% to10% by weight of the formulation.

The nebulizer formulation may also contain a surfactant to reduce orprevent surface induced aggregation of the protein caused by atomizationof the solution in forming the aerosol. Various conventional surfactantscan be employed, such as polyoxyethylene fatty acid esters and alcohols,and polyoxyethylene sorbitan fatty acid esters. Amounts will generallyrange between 0.001% and 4% by weight of the formulation. An especiallypreferred surfactant for purposes of this invention is polyoxyethylenesorbitan monooleate.

Specific formulations and methods of generating suitable dispersions ofliquid particles of the invention are described in WO 94/20069, U.S.Pat. No. 5,915,378, U.S. Pat. No. 5,960,792, U.S. Pat. No. 5,957,124,U.S. Pat. No. 5,934,272, U.S. Pat. No. 5,915,378, U.S. Pat. No.5,855,564, U.S. Pat. No. 5,826,570 and U.S. Pat. No. 5,522,385 which arehereby incorporated by reference.

Formulations for use with a metered dose inhaler device will generallycomprise a finely divided powder. This powder may be produced bylyophilizing and then milling a liquid conjugate formulation and mayalso contain a stabilizer such as human serum albumin (HSA). Typically,more than 0.5% (w/w) HSA is added. Additionally, one or more sugars orsugar alcohols may be added to the preparation if necessary. Examplesinclude lactose maltose, mannitol, sorbitol, sorbitose, trehalose,xylitol, and xylose. The amount added to the formulation can range fromabout 0.01 to 200% (w/w), preferably from approximately 1 to 50%, of theconjugate present. Such formulations are then lyophilized and milled tothe desired particle size.

The properly sized particles are then suspended in a propellant with theaid of a surfactant. The propellant may be any conventional materialemployed for this purpose, such as a chlorofluorocarbon, ahydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon,including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant. Thismixture is then loaded into the delivery device. An example of acommercially available metered dose inhaler suitable for use in thepresent invention is the Ventolin metered dose inhaler, manufactured byGlaxo Inc., Research Triangle Park, N.C., USA.

Formulations for powder inhalers will comprise a finely divided drypowder containing conjugate and may also include a bulking agent, suchas lactose, sorbitol, sucrose, or mannitol in amounts which facilitatedispersal of the powder from the device, e.g., 50% to 90% by weight ofthe formulation. The particles of the powder shall have aerodynamicproperties in the lung corresponding to particles with a density ofabout 1 g/cm² having a median diameter less than 10 micrometers,preferably between 0.5 and 5 micrometers, most preferably of between 1.5and 3.5 micrometers. An example of a powder inhaler suitable for use inaccordance with the teachings herein is the Spinhaler powder inhaler,manufactured by Fisons Corp., Bedford, Mass., USA.

The powders for these devices may be generated and/or delivered bymethods disclosed in U.S. Pat. No. 5,997,848, U.S. Pat. No. 5,993,783,U.S. Pat. No. 5,985,248, U.S. Pat. No. 5,976,574, U.S. Pat. No.5,922,354, U.S. Pat. No. 5,785,049 and U.S. Pat. No. 5,654,007.

Mechanical devices designed for pulmonary delivery of therapeuticproducts, include but are not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to those ofskill in the art. Specific examples of commercially available devicessuitable for the practice of this invention are the Ultravent nebulizer,manufactured by Mallinckrodt, Inc., St. Louis, Mo., USA; the Acorn IInebulizer, manufactured by Marquest Medical Products, Englewood, Co.,USA; the Ventolin metered dose inhaler, manufactured by Glaxo Inc.,Research Triangle Park, N.C., USA; the Spinhaler powder inhaler,manufactured by Fisons Corp., Bedford, Mass., USA the “standing cloud”device of Nektar Therapeutics, Inc., San Carlos, Calif., USA; the AIRinhaler manufactured by Alkermes, Cambridge, Mass., USA; and the AERxpulmonary drug delivery system manufactured by Aradigm Corporation,Hayward, Calif., USA.

Kits

The present invention also provides kits including the polypeptides,conjugates, polynucleotides, expression vectors, cells, methods,compositions, and systems, and apparatuses of the invention. Kits of theinvention optionally comprise at least one of the following of theinvention: (1) an apparatus, system, system component, or apparatuscomponent as described herein; (2) at least one kit component comprisinga polypeptide or conjugate or polynucleotide of the invention; a plasmidexpression vector encoding a polypeptide of the invention; a cellexpressing a polypeptide of the invention; or a composition comprisingat least one of any such component; (3) instructions for practicing anymethod described herein, including a therapeutic or prophylactic method,instructions for using any component identified in (2) or anycomposition of any such component; and/or instructions for operating anyapparatus, system or component described herein; (4) a container forholding said at least one such component or composition, and (5)packaging materials.

In a further aspect, the present invention provides for the use of anyapparatus, component, composition, or kit described above and herein,for the practice of any method or assay described herein, and/or for theuse of any apparatus, component, composition, or kit to practice anyassay or method described herein.

EXAMPLES

The following examples are offered to illustrate the present invention,but not to limit the spirit or scope of the present invention in anyway.

Materials and Methods

I. Determination of Surface-Accessible Residues

Accessible Surface Area (ASA)

The computer program Access (B. Lee and F. M. Richards, J. Mol. Biol.55: 379-400 (1971)) version 2 (© 1983 Yale University) was used tocompute the accessible surface area (ASA) of the individual atoms in thestructure. This method typically uses a probe-size of 1.4 Å and definesthe Accessible Surface Area (ASA) as the area formed by the center ofthe probe. Prior to this calculation all water molecules and allhydrogen atoms should be removed from the coordinate set, as shouldother atoms not directly related to the protein.

Fractional ASA of Side Chain

The fractional ASA of the side chain atoms is computed by division ofthe sum of the ASA of the atoms in the side chain by a valuerepresenting the ASA of the side chain atoms of that residue type in anextended ALA-x-ALA tripeptide. See Hubbard, Campbell & Thornton (1991)J. Mol. Biol. 220, 507-530. For this example the CA atom is regarded asa part of the side chain of glycine residues but not for the remainingresidues. The following values are used as standard 100% ASA for theside chain (Table 6): Ala 69.23 Å² Arg 200.35 Å² Asn 106.25 Å² Asp102.06 Å² Cys 96.69 Å² Gln 140.58 Å² Glu 134.61 Å² Gly 32.28 Å² His147.00 Å² Ile 137.91 Å² Leu 140.76 Å² Lys 162.50 Å² Met 156.08 Å² Phe163.90 Å² Pro 119.65 Å² Ser 78.16 Å² Thr 101.67 Å² Trp 210.89 Å² Tyr176.61 Å² Val 114.14 Å²

Residues not detected in the structure are defined as having 100%exposure as they are thought to reside in flexible regions. In the casewhere an ensemble of NMR structures is analyzed, the average ASA valueof the ensemble is used.

Determination of Surface Exposed Residues when no Three-DimensionalStructure is Available:

When no three-dimensional structure is available or if the structure isnot detailed enough to determine surface accessibility (e.g. if only theposition of the CA atoms is known) the surface accessibility may beinferred from a sequence alignment created as follows:

-   -   A: If the structure is known but not detailed enough to        determine surface accessibility:        -   The low detail structure is included in a structure-based            sequence alignment to the known structures of the sequence            family using the MODELER program available from Molecular            Simulations, Inc.    -   B: If no structure is known:        -   The sequence is aligned to a predefined sequence alignment,            including the sequences of the known structures of the            sequence family, that may be prepared using the            “profile/structure alignment” option of the program ClustalW            (Thompson et al. (1994) Nucleic Acids Research            22:4673-4680).

From the sequence alignment obtained in A or B, residues in the sequenceto be analyzed at positions equivalent to residues exposed in at leastone of the other sequences having a known structure are defined as beingexposed. The degree of exposure is taken to be the largest value for theequivalent residues in the other sequences. In cases where the sequenceto be analyzed is at an insertion (i.e. there are no equivalent residuesin the other sequences) this residue is defined as being fully exposed,as it most probably is located in a turn/loop region. In cases where alow detailed structure exists, those residues not observed in thestructure are defined as being fully exposed, as they are thought to bein flexible regions.

Determining Distances between Atoms:

The distance between atoms is readily determined using moleculargraphics software, e.g. InsightII® 98.0 from Molecular Simulations, Inc.

II. Protein Expression and Purification

A. Expression and Purification from CHO Cells

Some polypeptides of the invention were produced in Chinese HamsterOvary (CHO) K1 cells (ATCC: CCL-61) that were stably transfected andselected with G418 to establish clonal cell lines.

1. CHO Expression Construct:

Nucleic acids encoding polypeptides of the invention were cloned into aCHO expression vector, under control of the SV40 promoter and in-framewith a sequence which encodes an N-terminal leader sequence, and,optionally, one or two a C-terminal tag sequences. The leader sequencewas either a generic leader sequence, IFN alpha 6 leader or a modifiedIFN alpha 6 leader sequence, and C-terminal tags included an E-tag(Amersham Biosciences) &/or a His-tag. Plasmid production was inXL1-Blue cells.

2. Selection of Stable Subclones Expressing IFN-Alpha Polypeptides:

Materials:

Culture medium: DMEM-F12 with G418, FBS and Penicillin, Streptomycin andGlutamine (PSG; Gibco/Invitrogen);

1×PBS (Gibco/Invitrogen);

Trypsin/EDTA

Anti E-Tag Antibody-HRP conjugate (Amersham BioSciences)

ECL Plus Western Blotting detection Reagents (Amersham BioSciences)

Procedure: Stable transfectants were generated under selection with G418in DMEM/F-12 medium with FBS and penicillin. Cells were split into T175flasks with 50 ml of selection medium and incubated in a 37° C. CO₂incubator for ˜24 hr. or until cells reached 80% confluence. Cells wereharvested by washing with PBS followed by addition of 2.5 mlTrypsin/EDTA and incubation at 37° C. for 3-5 min. Cells were collectedand recovered by centrifugation at 1000 g for 30 min in a Beckman Modelbench top centrifuge. Cells were washed once in PBS and resuspended in 3ml PBS with 1% FBS. The cell density was determined and adjusted to1×10⁶ cell/ml with PBS/FBS. For each IFN-alpha, polypeptide cells weresorted in a DakoCytomation MoFlo sorter into 2-5 96 well platescontaining 200 ml of selection medium. The plates were incubated in a37° C. incubator for 10-14 days to allow the sorted cells to grow. Twosubclones were selected for each IFN-alpha polypeptide for high levelexpression first by dot blot analysis and subsequently confirmed byWestern blot analysis using an anti-E tag antibody-HRP conjugate andchemiluminescent detection.

3. Protein Expression:

Materials:

DMEM-F12 medium (Gibco/Invitrogen)

Ultra CHO medium (BioWhittaker)

CHO III A medium (Gibco/Invitrogen)

Ex-Cyte Growth Enhancing Media supplement (Serologicals Proteins)

ITSA (Insulin, Transferrin, Selenium supplement for adherent culture;Gibco/Invitrogen)

Penicillin/Streptomycin (P/S)

FBS, PBS, Trypsin

Procedure:

Day 1: Cells from one T-175 flask were transferred to one roller bottle(1700 cm²) in 300 ml DMEM-F12 with 10% FBS and 1×P/S and grown in a 37°C. CO₂ incubator.

Day 3: Medium was changed to 300 ml fresh DMEM-F12-FBS-P/S.

Day 5: The medium was changed to 300 ml Ultra CHO with 1/1000 Ex-Cyteand P/S.

Day 7: The media was replaced with 300 ml CHO III A+P/S productionmedium.

Supernatants were harvested on Day 8, 9 and 10. The supernatants werecentrifuged at 2000 g for 20 min in a Beckman Coulter Allegra 6R benchtop centrifuge and filtered using a 0.2μ PES bottle top sterile filterand stored at 4° C. for purification.

4. Protein Purification:

Some polypeptides of the invention were expressed as fusion proteinscontaining a 13 amino acid E-tag sequence at the C-terminus. Suchpolypeptides were purified using an E-tag affinity column, as follows.

Materials: Recombinant Phage Antibody System Purification Module(Amersham BioSciences, Cat. No. 17-1362-01). Purification kit contains a16 mm diameter×25 mm height (5 ml bed volume) anti E-Tag column andassociated buffers.

Procedure: Supernatants collected from CHO-HK1 cells in roller bottleswere clarified using a combination of centrifugation at 2800×g for 20min and filtration using a 0.2μ PES bottle top filter module.Supernatants were loaded onto the E-tag column equilibrated in RPASbinding buffer at 150 cm/h (5 ml/min). The column was washed with 5 CV(column volume) of binding buffer and the protein was eluted at 75 cm/h(2.5 ml/min) with RPAS elution buffer. Elution fractions wereneutralized with 0.05 volumes of 1M Tris-Cl pH 8.0, dialyzed into PBS,concentrated to 0.1-1.5 mg/ml and stored frozen in aliquots at −80° C.Samples for assays were formulated at 50 μg/ml in PBS with 0.5% BSA andstored frozen in aliquots at −80° C. Samples were routinely analyzed bySDS-PAGE followed by Coomassie staining using materials, reagents andprotocols obtained from Invitrogen.

Protein concentrations were routinely determined by the BCA assay usingan IFN-alpha standard, the concentration of which had been verified byamino acid analysis.

B. Expression and Purification from E. coli

Some polypeptides of the invention were produced in E. coli as inclusionbodies, which were purified and refolded as follows.

1a. E. coli Expression Construct

In some instances, nucleic acids encoding polypeptides of the inventionwere modified for improved expression in E. coli. Such modificationscomprised replacing rare Arg-Arg codon pairs AGGAGG at nucleotidepositions 34-39 and AGGAGA at nucleotide positions 64-69 (positionnumbering relative to SEQ ID NO:59) each with Arg-Arg codon pairs suchas CGTCGC which are preferred in E. coli, or replacing most or all rareArg codons AGA and AGG in the coding sequence with preferred E. coli Argcodons CGC or CGT. A methionine codon (ATG) was also added to the 5′ endof the coding sequence. Exemplary coding sequences modified in such amanner include SEQ ID NOs: 60-61, 63-64, 66-67, 69-70, 72-73, 75-76,78-79, 81-82, 84-85, and 87-88. The coding sequence was placed into thepET-42 expression vector (Novagen) under control of a T7 promoter withkanamycin selection marker, or into the pQE80-Kan expression vector(Qiagen) under the control of a T5 promoter with kanamycin selectionmarker.

1b. Protein Expression

pET-42 vectors containing interferon coding sequences were transformedinto an E. coli strain such as BL21(DE3) using standard methods, andplated on to agar plates containing 50 μg/ml kanamycin and incubated at37° C. After 18-24h, three separate colonies were picked and transferredinto tubes containing 5 ml of 2×YT with 50 μg/ml kanamycin and incubatedovernight at 37° C. The overnight culture was used to inoculate 2 setsof flasks containing 100 ml of 2×YT with 50 μg/ml kanamycin. The growthof the culture at 37° C. was monitored at OD₆₀₀. The culture was inducedat an OD₆₀₀ of 0.5-0.8 with 1 mM IPTG for 3 h at 37° C. IPTG inducedcultures were analyzed for expression by SDS-PAGE by lysing pelletedcells in SDS sample buffer. The corresponding uninduced sets of cultureswere used to prepare frozen stocks by addition of 25% glycerol andfreezing cells in 1 ml aliquots at −80° C. pQE80-Kan vectors containinginterferon coding sequences were transformed into E. coli strains W3110or W3110-fhuA. Expression was verified as described above.

Larger scale shake flask expression was performed by inoculating 4×1L2×YT media+kanamycin with 25 ml of an overnight culture. Cultures weremonitored at OD600 and induced with 1 mM IPTG at 0.5-0.8 OD units. After3 h of induction cells are harvested by centrifugation at 5000 g andstored frozen at −80° C. Cells were disrupted using 2-3 passes through aFrench press or a APV 1000 homogenizer at 10,000 psi and processed asdescribed under “Isolation of IB” and subsequent sections.

Fed-batch fermentation was conducted at 10 L scale in a B. Braunbioreactor in Terrific Broth (TB) medium supplemented with trace elementsolution and 40 mg/L kanamycin. Fermentation was initiated byinoculating the bioreactor with a 400 ml overnight culture in TB medium.During the initial growth phase the dissolved oxygen (DO) was maintainedat 50% by varying the agitation rate. When the OD600 of the culturereached 5.0, the glycerol/amino acid feed was initiated at 0.5 ml/minand the agitation was set to 1000 rpm. The feed rate was adjusted tomaintain the DO at 40% for the rest of the fermentation process. Whenthe OD600 reached 25 the culture was induced by addition of IPTG to afinal concentration of 1 mM. Three hours post induction the cells wereharvested by centrifugation at 10000×g in a Beckman centrifuge and thecell paste was stored at −60° C. The OD600 at harvest was typicallyaround 35-40.

2a. E. coli Expression Construct (for High-Level Expression)

The following example shows the expression of two exemplary polypeptidesof the invention cloned into an E. coli expression system (as describedin EP 0972838) allowing high level expression of eukaryotic genes in E.coli. Coding sequences corresponding to the amino acid sequences of14epi18 (SEQ ID NO:13) and 25epi19 (SEQ ID NO:38), both containing anadditional Met at the N-terminus, were optimized according tocodon-usage and mRNA secondary structure (Griswold et al., Protein ExprPurif. 2003 January; 27(1):134-42) using the sequence analysis programGCG (Genetic Computer Group, Inc., Wisconsin USA, Version 7.1, March1992) function “backtranslate”/“EcoHigh-database” to prevent rarecodons, and function MFold/Plotfold (-menu=f or -menu=g) for predictionand prevention of potential secondary structures detrimental to the geneexpression. Coding sequences optimized in this manner are provided asSEQ ID:89 and SEQ ID NO:90, respectively.

The optimized DNA sequences were cloned into OripBR-URA3 with anadditional lacI gene. The resulting vectors, designatedOripBR-URA3-lacI_(—)14epi18-mut2 (FIG. 6) andOripBR-URA3-lacI_(—)25epi19-mut (FIG. 7), were transformed intocompetent E. coli UT5600 (ΔpyrF) cells (as described in EP 0972838).Transformed cells were selected on minimal M9-medium agar plates withoutaddition of Uracil.

M9-Agar plates: Sterile agarose stock solution (final conc. 15 g/l) 430ml M9-salt solution 50 ml 1M MgSO₄ 0.5 ml 1M CaCl₂ 0.05 ml 20% Glucose 5ml Vitamin B1 (c = 1 mg/ml) 2.5 ml 10% Casaminoacids 25 ml L-Tryptophan(c = 5 mg/ml) 5 ml L-Leucine (c = 5 mg/ml) 5 ml L-Proline (c = 5 mg/ml)2.5 ml

2b. Protein Expression

Transformed cells according to 2a. above were inoculated in shake flasksand cultured at 37° C. Induction was performed at OD₅₇₈=1 by addition of1 mM IPTG. Cells were harvested after 7 h and cell pellets were analyzedon an SDS-PAGE gel. The media used is as follows: Casaminoacids 120 gNH₄Cl 3.9 g Tryptophan 5.5 g Leucine 5.5 g Proline 5.5 g Glycerin 85 gdissolved in 4300 ml H₂O and K₂HPO₄*3H₂O 91.5 g Citric acid 8.0 gdissolved in 600 ml H2O and Thiamin (5 mg/ml) 17.5 ml MgSO₄ × 7H₂O (1M)17.5 ml pH = 7.0 adjust to 5000 ml with H₂O

3. Isolation, Solubilization, Sulfonation and Refolding of InclusionBodies (IB)

For isolating IB the thawed cell paste was resuspended in 1×PBS at 10 mlper gram of cell paste and mixed until a uniform slurry was obtained.The cells were disrupted by two passes through a microfluidizer at17,000-19,000 psi. The cell lysate was adjusted to 1% Triton-X100, mixedfor 10 min and the IB were recovered by centrifugation at 10,000 g for60 min at 2-8° C. The IB pellet was washed once by resuspending in PBSwith 1% Triton-X100 and recovered by centrifugation as above and storedfrozen at −80° C.

3a. Solubilization/Refolding Method 1:

For solubilizing IB, the IB pellet was resuspended in Urea buffer (50 mMTris-Cl, 200 mM NaCl, 8 M urea, 2 mM DTT, 1 mM EDTA, pH 8.0) at 10 mlbuffer per gram of cell paste. The suspension was mixed for 30 min andcentrifuged at 10,000 g for 30 min at 2-8° C. The pellet was washed onceby resuspending in the Urea buffer without DTT, centrifuged as above andwashed twice with water. The washed pellet was solubilized in Guanidinebuffer (50 mM Tris-Cl, 200 mM NaCl, 8 M guanidine-HCl, 1 mM EDTA, pH8.0) at 10 ml buffer per gram pellet, mixed for 30-60 min andcentrifuged at 10,000 g for 30 min at 2-8° C. The supernatant containingthe solubilized IFN was adjusted to 10 mg/ml sodium sulfite and 5 mg/mlsodium tetrathionate to initiate the sulfonation process which wasperformed at 2-8° C. for 16 hours. Post-sulfonation the IFN solution wasdiluted 2-fold with water and the IFN pellet was recovered bycentrifugation at 10,000 g at 2-8° C. The pellet was washed twice withwater and resuspended in Guanidine buffer as above. The proteinconcentration of the sulfonated IFN was determined by absorbance at 280nm.

The refolding process was initiated by diluting the sulfonated IFN inGuanidine buffer at 2-8° C. to a final concentration of 100 mg/ml inRefolding buffer (50 mM Tris-Cl, 20 mM NaCl, 2 mM reduced glutathione, 1mM oxidized glutathione). Refolding was performed at 2-8° C. with slowmixing for 6-8 h, followed by addition of CuSO₄ to a final concentrationof 2 μM followed by an additional refolding period of 16-20 h. Theprogress of the refolding reaction was monitored by SDS-PAGE and reversephase HPLC.

3b. Solubilization/Refolding Method 2:

For solubilization of 14epi18, the IB pellet was resuspended inGuanidine buffer (7 M Guanidine, 20 mM Na₂HPO₄, 10 mM DTE, pH 8.0) at 4ml buffer per gram of IB paste. The suspension was mixed for 2 hoursand, after solubilization, diafiltered at least 8 volumes at roomtemperature against a buffer containing 7 M Guanidine buffer and 20 mMNaH₂PO₄ pH 5.0 using 10 kDa cut-off membranes.

For refolding, the solubilized protein was diluted into a folding buffercontaining 20 mM Na₂HPO₄, 0.8 M arginine and 2 μM CuSO₄, pH 7.5.Refolding was performed at 2-8° C. with slow mixing for 72 hours at afinal protein concentration of about 0.5 mg/ml protein. The progress ofthe refolding reaction was monitored by reverse phase HPLC.

3b. Solubilization/Refolding Method 3:

For solubilization of 25epi19, the IB slurry was resuspended insolubilisation buffer (8 M Guanidinium chloride, 100 mM DTT, 50 mM TrispH 8.3). The suspension was mixed for 30 minutes at room temperature.Subsequently the pH was adjusted to 4.5 with HCl. The solubilisate wasfurther concentrated using a 5 K membrane and diafiltrated against a 10fold volume of 7.2 M Guanidinium chloride adjusted to pH 4.5 with HCl.

For refolding, the solubilized protein was diluted into a folding buffer(0.8 M Arginine, 50 mM potassium phosphate, 2 mM GSH, 1 mM GSSG pH 7.8).Refolding was performed at 2-8° C. overnight at a final concentration ofabout 1 mg/ml protein.

4. Purification

4a. Purification Method 1:

The refolded IFN was purified using three chromatography steps. Therefolding solution was adjusted to 80% saturating concentration ofammonium sulfate (weight/volume), filtered through a 0.2 μM filter andloaded at 200 cm/h onto a 20 ml Butyl Sepharose Fast Flow HydrophobicInteraction Chromatography (HIC) column (Amersham Biosciences)equilibrated in Equil buffer (50 mM Tris-Cl, 0.8 M ammonium sulfate, pH8.0). The HIC column was washed with 8 column volumes (CV) of Equilbuffer, followed by 8 CV of Wash buffer (50 mM Tris-Cl, 0.5 M ammoniumsulfate, pH 8.0). IFN was eluted with 50 mM Tris-Cl, pH 8.0 with orwithout 15% saturating concentration of ammonium sulfate.

The HIC pool was adjusted to 50 mM sodium acetate using 0.5 M sodiumacetate, pH 4.5 stock and diluted two fold. This adjusted pool wasloaded at 90 cm/h on to a 5 ml HiTrap CM Sepharose Fast Flow column(Amersham Biosciences), equilibrated in 50 mM sodium acetate, 100 mMNaCl, pH 5.0. Post loading the column was washed with 5 CV underequilibration buffer conditions and IFN was eluted using a 20 CVgradient from 100-650 mM NaCl. Peak fractions containing IFN were pooledbased on absorbance at 280 nm.

The CM Sepharose pool was adjusted to 20 mM 1,3 diaminopropane using a 2M 1,3 diaminopropane stock to set pH at ˜10. The sample was loaded at200 cm/h on to a 5 ml HiTrap Q Sepharose Fast Flow column (AmershamBiosciences), equilibrated in 50 mM 1,3 diaminopropane, 100 mM NaCl, pH10.0. Post loading the column was washed with 5 CV of buffer underequilibration conditions and IFN was eluted using a 20 CV gradient from100-650 mM NaCl or from 100-1000 mM NaCl. The fractions containing IFNare pooled based on absorbance at 280 nm and immediately dialyzedagainst 250-500 volumes of 50 mM sodium acetate, 150 mM NaCl, pH 5.0 or50 mM sodium borate, pH 9.0. Post-dialysis the IFN samples were sterilefiltered using a 0.2 μM filter in a biosafety cabinet, the concentrationwas measured by absorbance at 280 nm and the samples are stored at 2-8°C. for periods up to a week or in aliquots at −80° C. for extendedstorage. For activity assays the sample was generally formulated in 0.5%BSA, PBS pH 7.4 at 5, 20 and 50 ug/ml, and stored frozen in aliquots at−80° C.

4b. Purification Method 2:

Refolded 14epi18 was purified using two chromatographic steps. Therefolding solution was adjusted to pH 8.0 and around 190 mS/cm with KCl,filtered through a 5 μm filter and loaded at 75 cm/h onto aButyl-Sepharose Fast Flow Hydrophobic Interaction Chromatography (HIC)column (General Electric Healthcare) equilibrated in equilibrationbuffer consisting of 2.0 M KCl, 20 mM K₂HPO₄, 0.8 M arginine, pH 8.0.The HIC column was washed with 5 column volumes (CV) of 2.0 M KCl, 20 mMK₂HPO₄, 0.8 M arginine pH 8.0. 14epi18 was eluted with 0.4 M KCl, 20 mMK₂HPO₄, pH 8.0. The elution was monitored by reverse phase HPLC.

The eluate was diluted with distilled water to a conductivity of around3 mS/cm. The pH was adjusted to 6.8. This adjusted pool was loaded at 50cm/h onto a Q-Sepharose Fast Flow column (GE Healthcare) equilibrated in25 mM ammonium acetate pH 6.8. Post loading, the column was washed withequilibration buffer. 14epi18 was eluted with a linear gradient to pH4.0. Peak fractions containing the folded 14epi18 were pooled based onreversed phase HPLC analytics.

4c. Purification Method 3:

Refolded 25epi19 was purified using two chromatographic steps. Therefolding solution was adjusted to 1.3 M NaCl and the solution wasfiltered over a combination of two filters (0.8/0.3 μm followed by0.45/0.2 μm). The protein was loaded onto a Butyl Sepharose Fast Flowcolumn (General Electric Healthcare) equilibrated with equilibrationbuffer (0.8 M Arginine, 1.3 M sodium chloride, 50 mM potassium phosphatepH 8.0) at 50-150 cm/h. After sample application, the column was washedwith 3 CV equilibration buffer and 3 CV wash buffer (20 mM potassiumphosphate, 2 M sodium chloride pH 7.5). Step elution was performed byapplying elution buffer (20 mM potassium phosphate, pH 7.5) with a flowof 50-100 cm/h. The peak was pooled according to UV280 measurement.

After assuring that the conductivity was below 10 mS/cm, the pool wasloaded at 50-75 cm/h onto a Q-Sepharose Fast Flow column (GE Healthcare)equilibrated in 30 mM ammonium acetate, pH 5.9. Post loading, the columnwas washed with equilibration buffer until baseline (UV280) was reached.25epi19 was eluted with a linear gradient to pH 3.5. Peak fractionscontaining the folded 25epi19 were pooled according to reversed phaseHPLC analytics and SDS-PAGE.

III. Activity Assays

A1. HuH7-EMCV Antiviral Assay

Provided below is an exemplary assay for antiviral activity ofinterferon-alpha polypeptides and conjugates of the invention. The assayis a cell-based dose-response assay used to assess the anti-viralpotency of a drug, and is sometimes referred to as “protection fromcytopathic effect” (or PCPE) assay, also termed inhibition of cytopathiceffect (CPE) assay. Briefly, cells are incubated with drug and exposedto virus. In the absense of drug, cells exposed to virus die. Withincreasing concentrations of drug, an increasing proportion of cellssurvive. The number of surviving cells can be measured directly (e.g.,by visual counts) or indirectly by estimating metabolic rate. Forexample, metabolic dyes such as MTT or WST-1 may be used as an indirectmeasure of cell survival. Live cells metabolize such dyes to formmetabolic products which can be quantified by spectrophotometry (opticaldensity).

Materials and Reagents:

HuH7 Cells: Human hepatoma cell line (obtained from Dr. Michael Lai,USC-Surgery Department, Los Angeles, Calif.). The cell line may also beobtained from the Cell Bank of the Japanese Collection of ResearchBioresources (JCRB)/Health Science Research Resources Bank (HSRRB),Osaka, Japan. The HuH7 cell line was originally established in thelaboratory of Dr. J. Sato (Okayama University School of Medicine) from a57-year old Japanese male with well-differentiated hepatocellularcarcinoma (Nakabayashi, H., et al. (1982) Cancer Res. 42(9):3858-63).The cell line is negative for Hepatitis B surface antigen.

VERO Cells: African green monkey kidney cell line (ATCC # CCL-81). Thecell line was derived in 1962 at Chiba University in Chiba, Japan fromthe kidney of a normal adult African green monkey. Passage # 93 of theVERO cell line was transferred to the National Institute of Health (NIH)in 1964.

Encephalomyocarditis virus (EMCV): tissue culture adapted strain (ATCC #VR-129B). A tissue culture adapted strain of encephalomyocarditis virus(EMCV) was purchased from the ATCC (# VR-129B). For the HuH7 antiviralassays, a high titer viral stock was produced in Vero African greenmonkey kidney cells. The viral titer of the Vero produced EMCV stock wasdetermined by plaque assay on L929 cells to be 1.1×10⁸ PFU/ml.

Complete DMEM:

-   -   Dulbecco's Modified Eagle Medium (DMEM, Gibco Cat. No.        11965-092)    -   10% Fetal Bovine Serum (FBS, Hyclone Cat. No. SH30071.03)    -   1× Penicillin-streptomycin (PS, Gibco Cat. No. 15140-122)

Reduced Serum DMEM:

-   -   Dulbecco's Modified Eagle Medium (DMEM, Gibco Cat. No.        11965-092)    -   2% Fetal Bovine Serum (FBS, Hyclone Cat. No. SH30071.03)    -   1× Penicillin-streptomycin (PS, Gibco Cat. No. 15140-122)

Trypsin/EDTA (Gibco Cat. No. 25300-054)

WST-1 (Roche; Cat. No. 1 644 807)

HuH7 cells were maintained in Complete DMEM at 37° C. in a humidified 5%CO₂ incubator. The cells were harvested with trypsin and split twiceweekly when confluent to a final density of 1-2×10⁶ cells per 25 ml in aT175 flask. One day prior to the assay, the cells were trypsinized andseeded into new T175 flasks at a density of 4.5×10⁶ cells per 25 ml toensure that the cells were in log phase prior to the assay.

HuH7-EMCV Antiviral Assay Procedure:

A high titer EMCV virus stock was amplified in VERO cells. The lethalconcentration at which 95% of the cells were killed (LC₉₅) wasdetermined by an EMCV viral killing curve on HuH7 cell monolayers.Briefly, HuH7 cells were plated on day one in 96-well microtiter platesat 6×10⁴ cells per well. Virus was serial diluted 1:3 in DMEM+2% FBSwith 10 dilution points and added to the cells on day two. Twenty-fourhours post-infection, cell survival was determined by a tetrazolium saltmetabolism assay, WST-1 (Roche). The LC₉₅ determined for the HuH7-EMCVassay corresponded to an MOI of 0.034 (PFU/cell). Titer of the virusstock was determined by a standard plaque assay on L929 cells.

On day one of the assay, log phase HuH7 cells were harvested withtrypsin, resuspended in Reduced Serum DMEM and concentrated bycentrifugation. The cell pellets, corresponding to 5 T175 flasks ofcells, were resuspended in 10 ml of Reduced Serum DMEM, filtered througha 40 micron Nylon cell strainer and counted with a hemocytometer. Cellviability was determined by trypan blue exclusion. The cells wereresuspended in Reduced Serum DMEM to a final density of 6×10⁵ cells/ml.One hundred microliters of the diluted cells were added to each well ofa 96-well assay plates (6×10⁴ cells/well) and the plates were incubatedat 37° C. in a humidified 5% CO₂ incubator for 4 hours.

The potencies of “reference” interferon alphas and interferon-alphapolypeptides of the invention (also called “test samples”) weredetermined by dose-response analysis. There was generally one testsample and one reference IFN-alpha per plate, each with three replicatecurves of IFN-alpha treated/EMCV challenged cells and two replicatecurves of IFN-alpha treatment alone. The later was assayed to controlfor potential antiproliferative effects of IFN-alpha on HuH7 cells. Thedose-response curves for the reference IFN-alphas generally consisted of8 three-fold dilutions ranging from 100 ng/ml to 0.05 ng/ml. For theIFN-alpha test samples, the three-fold dilutions generally ranged from 5ng/ml to 0.002 ng/ml. Eight wells each of cells treated with virus butno IFN-alpha and cells alone were also run as controls.

The IFN-alpha dilutions were prepared using Reduced Serum DMEM. Onehundred microliters of the diluted IFN-alpha preparations weretransferred to the assay plates. The assay plates were incubated at 37°C. in a humidified 5% CO₂ incubator for 16 hours.

On day two, the cells were challenged with EMC virus. Medium wasaspirated from each well of the assay plate. The EMCV stock was diluted1:5400 in DMEM+2% FBS. One hundred microliters of the diluted virus,corresponding to 0.034 viral particles per cell initially seeded, wasadded to each well. The cells were incubated with virus at 37° C. in ahumidified 5% CO₂ incubator for 24 hours.

On day three, the number of viable cells in each well was quantified byWST-1 assay. Medium was aspirated from each well of the assay plate. TheWST-1 reagent was diluted 1:20 in Reduced Serum DMEM, 100 μl of thediluted WST-1 reagent was added to each well and the cells wereincubated at 37° C. in a humidified 5% CO₂ incubator for 60 minutes. Thenumber of viable cells in each well was quantified by measuring OD at450 nm on a plate reader.

Analysis:

The antiviral potency of the IFN-alpha reference and test samples werecalculated with the equation:Antiviral potency=(Viable cells_(C+I+V)−Viable cells_(C+V))/(Viablecells_(C+I)−Viable cells_(C+V))*100%

where C+V=HuH7 cells+EMCV, C+I=HuH7 cells+IFN-α, and C+I+V=HuH7cells+IFN-α+EMCV

Dose-response curves were analyzed by non-linear regression usingGraphPad Prism 4 (GraphPad Software Inc.) The following equation wasused for the curve fits:$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{1 + 10^{{({{{Log}\quad{EC}\quad 50} - X})} \cdot {HillSlope}}}}$

Bottom is the Y value at the bottom plateau; Top is the Y value at thetop plateau, and Log EC50 is the X value when the response is halfwaybetween Bottom and Top. The Levenberg-Marquardt method was used asoptimization algorithm.

A2. HeLa-EMCV and WISH-EMCV Antiviral Assays

The inhibition of the cytopathic effect of EMCV on HeLa (a humancervical carcinoma derived cell line) and WISH (a human amniotic tissuederived cell line) cells also provide useful surrogate assays to assessthe antiviral potencies of interferon alpha polypeptides and conjugatesof the invention. These assays were performed in a similar fashion tothe HuH7-EMCV assay described above, with the following modifications.

Materials and Reagents:

HeLa Cells: Human cervical carcinoma cell line (ATCC # CCL-2). The HeLacell line was derived from a cervical adenocarcinoma from a 35-year-oldfemale. The HeLa cells are reported to contain human papilloma virus(HPV-18) sequences. The cell line was received from the ATCC at passagenumber 107.

WISH Cells: Human epithelial cell line (ATCC # CCL-25). The WISH cellline was established from altered colonies appearing in a subculture ofprimary human amniotic cells after 35 days in culture. The WISH cellline contains HeLa marker chromosomes. The cells were received from theATCC at passage # 170.

L-929 Cells: Murine fibroblast cell line (ATCC # CCL-1). The L-929 cellline is a single cell clone isolated from the95^(th passage of the parental strain, Strain L, in) 1948. Strain L wasisolated from the subcutaneous areolar and adipose tissue of a normal100 day old male C[3]H/An mouse.

EMCV: tissue culture adapted strain (ATCC # VR-129B). A tissue cultureadapted strain of encephalomyocarditis virus (EMCV) was purchased fromthe ATCC (# VR-129B). For the HeLa and WISH antiviral assays, the hightiter EMCV stock was produced in L929 cells. The viral titer of the L929produced viral stock was 4.0×10⁸ PFU/ml based on the results of a plaqueassay on L929 cells.

Complete MEM:

-   -   Minimal Essential Media (MEM, Gibco Cat. No. 10370-021)    -   10% Fetal Bovine Serum (FBS, Hyclone Cat. No. SH30071.03)    -   1× Penicillin-streptomycin-glutamine (PSG, Gibco Cat. No.        10378-016)    -   1 mM Sodium pyruvate (Gibco Cat. No. #11360-070)

Reduced Serum MEM:

-   -   Minimal Essential Media (MEM, Gibco Cat. No. 10370-021)    -   2% Fetal Bovine Serum (FBS, Hyclone Cat. No. SH30071.03)    -   1× Penicillin-streptomycin-gluatimine (PSG, Gibco Cat. No.        10378-016)    -   1 mM Sodium pyruvate (Gibco Cat. No. #11360-070)

HeLa cells were maintained in Complete MEM at 37° C. in a humidified 5%CO₂ incubator. The cells were harvested with trypsin and split twiceweekly when confluent to a final density of 2-4×10⁶ cells per 25 ml in aT175 flask. One day prior to the assay, the cells were trypsinized andseeded into new T175 flasks at a density of 6×10⁶ cells per 25 ml toensure that the cells were in log phase prior to the assay.

WISH cells were maintained in Complete MEM at 37° C. in a humidified 5%CO₂ incubator. The cells were harvested with trypsin and split twiceweekly when confluent to a final density of 5-6×10⁶ cells per 25 ml in aT175 flask. One day prior to the assay, the cells were trypsinized andseeded into new T175 flasks at a density of 10×10⁶ cells per 25 ml toensure that the cells were in log phase prior to the assay.

HeLa-EMCV Antiviral Assay Procedure:

On day one of the assay, log phase HeLa cells were harvested withtrypsin, resuspended in Reduced Serum MEM and concentrated bycentrifugation. The cell pellets, corresponding to 5 T175 flasks ofcells, were resuspended in 10 ml of Reduced Serum MEM, filtered througha 40 micron Nylon cell strainer and counted with a Coulter Counter. Cellviability was determined by trypan blue exclusion with a hemocytometer.The cells were resuspended in Reduced Serum MEM to a final density of1×10⁵ cells/ml. One hundred microliters of the diluted cells were addedto each well of a 96-well assay plates (1×10⁴ cells/well) and the plateswere incubated at 37° C. in a humidified 5% CO₂ incubator for 24 hours.

The potencies of “reference” interferon alphas and interferon-alphapolypeptides of the invention (also called “test samples”) weredetermined by dose-response analysis. There was generally one testsample and one reference IFN-alpha per plate, each with three replicatecurves of IFN-alpha treated/EMCV challenged cells and two replicatecurves of IFN-alpha treatment alone. The later was assayed to controlfor potential antiproliferative effects of IFN-alpha on HeLa cells. Thedose-response curves for the reference IFN-alphas generally consisted of8 five-fold dilutions ranging from 60 ng/ml to 0.0008 ng/ml. For theIFN-alpha test samples, the five-fold dilutions generally ranged from 5ng/ml to 0.00006 ng/ml. Eight wells each of cells treated with virus butno IFN-alpha and cells alone were also run as controls.

The IFN-alpha dilutions were prepared using Reduced Serum MEM. Onehundred microliters of the diluted IFN-alpha preparations weretransferred to the assay plates. The assay plates were incubated at 37°C. in a humidified 5% CO₂ incubator for 16 hours.

On day three, the cells were challenged with EMC virus. Medium wasaspirated from each well of the assay plate. The EMCV stock was diluted1:133 in MEM+2% FBS. One hundred microliters of the diluted virus,corresponding to 3×10⁵ viral particles was added to each well. The cellswere incubated with virus at 37° C. in a humidified 5% CO₂ incubator for24 hours.

On day four, the number of viable cells in each well was quantified byWST-1 assay. Medium was aspirated from each well of the assay plate. TheWST-1 reagent was diluted 1:20 in Reduced Serum MEM, 100 μl of thediluted WST-1 reagent was added to each well and the cells wereincubated at 37° C. in a humidified 5% CO₂ incubator for 60 minutes. Thenumber of viable cells in each well was quantified by measuring OD at450 nm on a plate reader.

WISH-EMCV Antiviral Assay Procedure:

On day one of the assay, log phase WISH cells were harvested withtrypsin, resuspended in Reduced Serum MEM and concentrated bycentrifugation. The cell pellets, corresponding to five T175 flasks ofcells, were resuspended in 10 ml of Reduced Serum MEM, filtered througha 40 micron Nylon cell strainer and counted with a Coulter Counter. Cellviability was determined by trypan blue exclusion with a hemocytometer.The cells were resuspended in Reduced Serum MEM to a final density of3×10⁵ cells/ml. One hundred microliters of the diluted cells were addedto each well of a 96-well assay plates (3×10⁴ cells/well) and the plateswere incubated at 37° C. in a humidified 5% CO₂ incubator for 4 hours.

The potencies of “reference” interferon alphas and interferon-alphapolypeptides of the invention (also called “test samples”) weredetermined by dose-response analysis. There was generally one testsample and one reference IFN-alpha per plate, each with three replicatecurves of IFN-alpha treated/EMCV challenged cells and two replicatecurves of IFN-alpha treatment alone. The later was assayed to controlfor potential antiproliferative effects of IFN-alpha on HeLa cells. Thedose-response curves for the reference IFN-alphas generally consisted of8 ten-fold dilutions ranging from 100 ng/ml to 0.0001 ng/ml. For theIFN-alpha test samples, the five-fold dilutions generally ranged from 5ng/ml to 0.00006 ng/ml. Eight wells each of cells treated with virus butno IFN-alpha and cells alone were also run as controls.

The IFN-alpha dilutions were prepared using Reduced Serum MEM. Onehundred microliters of the diluted IFN-alpha preparations weretransferred to the assay plates. The assay plates were incubated at 37°C. in a humidified 5% CO₂ incubator for 16 hours.

On day two, the cells were challenged with EMC virus. Medium wasaspirated from each well of the assay plate. The EMCV stock was diluted1:267 in MEM+2% FBS. One hundred microliters of the diluted virus,corresponding to an MOI of approximately 5 was added to each well. Thecells were incubated with virus at 37° C. in a humidified 5% CO₂incubator for 24 hours.

On day three, the number of viable cells in each well was quantified byWST-1 assay. Medium was aspirated from each well of the assay plate. TheWST-1 reagent was diluted 1:20 in Reduced Serum MEM, 100 μl of thediluted WST-1 reagent was added to each well and the cells wereincubated at 37° C. in a humidified 5% CO₂ incubator for 60 minutes. Thenumber of viable cells in each well was quantified by measuring OD at450 nm on a plate reader.

B. T_(H)1 Differentiation Assay

Provided below is an exemplary assay for T_(H)1 differentiation activityof interferon-alpha polypeptides and conjugates of the invention.

Assay Procedure:

Human buffy coats (25-30 ml) containing leukocytes and erythrocytesprepared from 500 ml blood were collected from Stanford Blood Bank theday of assay initiation (one day before the assay day) and kept at roomtemperature overnight. Each buffy coat was carefully transferred to aT75 flask and diluted to 100 ml with PBS. For each buffy coat, 13 ml ofHistopaque/Ficoll (Sigma H8889) was pipetted into four 50 ml centrifugetubes, and 25 ml of diluted blood sample was carefully overlaid on topof the Histopaque/Ficoll without disrupting the interface. The tubeswere then centrifuged (20° C., 2500 rpm) for 20 minutes. Using 3 mlplastic transfer pipettes the mononuclear cell layer containing PBMCswas transferred to two 50 ml conical tubes (cells from 2Histopaque/Ficoll/buffy coat tubes to one tube). The PBMCs were thendiluted to 50 ml/tube with PBS and centrifuged (20° C., 1000 rpm) for 10minutes to remove platelets. After removal of PBS, the PBMCs andremaining RBCs were mixed to prevent aggregation. Ten milliliters of RBClysis buffer (ammonium chloride buffer) were added and two tubes ofcells were combined to one tube. Each tube, now containing the totalPBMC and RBC isolate from one donor, was incubated at room temperaturefor 10 min. Potential clots of blood cells were removed by filtering thecells with a cell strainer (70 um, Falcon Cat. No. 2350). PBS was addedto a total volume of 50 ml followed by centrifugation (20° C., 1000 rpm)for 10 min. The cell number was finally counted using a hemocytometer.

Next, a fraction of each PBMC preparation was prestained and analyzed byFACS to select PBMC preparations with a percentage of naïve THO cellsabove 15%. Three hundred microliters of PBMCs were stained with 20 μlFITC-conjugated anti-human CD45RA (Pharmigen, Cat. No. 555488), 20 μlCy-chrome conjugated anti-human CD4 (Pharmigen, Cat. No. 555348), 5 μlPE-conjugated anti-human CD8 (Pharmigen, Cat. No. 555367), 5 μlPE-conjugated anti-human CD14 (Pharmigen, Cat. No. 555398), and 5 μlPE-conjugated anti-human CD20 (Pharmigen, Cat. No. 555623), andincubated on ice for 45 minutes. The cells were washed with PBS,resuspended in 1 ml PBS, and filtered with a 40 μm cell strainer(Falcon, Cat. No. 2340). The percentage of naïve T_(H)0 cells (positiveto CD4 and CD45RA and negative to CD8, CD14, CD20) were quantified byFACS, and PBMC preparations with more than 15% naïve T_(H)0 cells wereselected for the assay.

The selected PBMC preparations were stained with 500 μl FITC-conjugatedanti-human CD45RA, 500 μl Cy-chrome conjugated anti-human CD4, 200 μlPE-conjugated anti-human CD8, 50 μl PE-conjugated anti-human CD14, and50 μl PE-conjugated anti-human CD20, and incubated on ice for 60minutes. The cells were washed with PBS, PI was added, and the cellswere diluted with 10 ml of PBS followed by filtering with a 40 μm cellstrainer. The cells were FACS sorted, and 1×10⁴ naïve T_(H)0 cells(positive to CD4 and CD45RA and negative to CD8, CD14, CD20) weretransferred by MOFLO into each well of 96 well round bottom plates,containing 160 μl DMEM plus Penicillin-streptomycin plus 2 mM Glutamineand 10% Fetal Bovine Serum (Hyclone Cat. No. SH30071.03).

One point seven microliters of Dynabeads CD3/CD28 T cell expander(Dynal, Cat. No. 111.32) were added to each well. The stimulatory effectof the Dynabeads was calibrated prior to the experiment to avoidlot-to-lot variance. Next, 20 μl/well of protein samples were added tothe assay plates. Generally, concentration ranges for IL-4 and IL-12standards (obtained from R&D Systems) were from 0.04 pg/ml to 10 ng/ml,and concentration ranges for IFN-alpha test samples and IFN-alphareference sample were from 0.76 pg/ml to 200 ng/ml.

The cells were incubated at 37° C. in a humidified 5% CO₂ incubator for6 days. Supernatants from each well were harvested to determine thedegree of T_(H)1 differentiation through quantification of the IFN-γcontent, using a standard ELISA (R&D Systems, Cat. # DIF50).

Analysis:

Response=IFN-γ concentrations in pg/ml .

The following equation was used for curve fitting:$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{1 + 10^{{({{{Log}\quad{EC}\quad 50} - X})} \cdot {HillSlope}}}}$

The variable Bottom is the Y value at the bottom plateau; Top is the Yvalue at the top plateau, and Log EC50 is the X value when the responseis halfway between Bottom and Top. The Levenberg-Marquardt method wasused as optimization algorithm.

C. Daudi Antiproliferation Assay

Provided below is an exemplary assay for antiproliferative activity ofinterferon-alpha polypeptides and conjugates of the invention.

Materials:

Daudi Cells: Human Burkitt's lymphoma cells (ATCC Number: # CCL-213).The Daudi line was derived from a 16-year-old black male with Burkitt'slymphoma in 1967. The cell line is positive for Epstein-Barr virus.

Complete RPMI:

-   -   RPMI 1640 (RPMI, Gibco Cat. No. 11875-143)    -   10% Fetal Bovine Serum (FBS, Hyclone Cat. No. SH30071.03)    -   1×Penicillin-streptomycin-gluatimine (PSG, Gibco Cat. No.        10378-01

Daudi Burkitt's lymphoma cells grown in suspension were maintained inT175 tissue culture flasks, containing 50 ml of complete RPMI at 37° C.in a humidified 5% CO₂ incubator. The cells were split 1:10 whenconfluent.

Assay Procedure:

Daudi cells were spun down and washed with 1×PBS. The cell number wasadjusted to 10⁵ cells/ml. 80 μl culture medium was added to each well in96 well round bottom assay plates followed by transfer of 100 μl cells(10⁴ cells/well) to each well.

Eleven dilutions of the IFN-alpha reference material and IFN-alpha testsamples, ranging from 200 ng/ml to 0.2 pg/ml (4-fold dilutions), wereprepared in dilution plates using culture medium. Twenty μl of thediluted IFN-alpha preparations were then transferred to the assayplates.

The cells were incubated at 37° C. in a humidified 5% CO₂ incubator.After 48 hours, 1 μCi of methyl-³H thymidine (Amersham Pharmacia, Cat.No. TRK758) was added to each well followed by incubation for 24 hoursat 37° C. in a humidified 5% CO₂ incubator. The cells were harvested onthe following day and incorporation of thymidine was determined.

Alternatively, the Daudi antiproliferation assay may be performed bymeasuring the number of viable, metabolically active Daudi cells basedon the amount of ATP present in a culture well following interferontreatment. In this procedure, 8000 Daudi cells are plated in a volume of50 μl in a 96 well round bottom assay plate. The cells are allowed toincubate at 37° C. for 2 hours in a 5% CO₂ incubator prior to theaddition of the interferon dilutions. The interferons are diluted 6-fold(9-points) generally starting from 5-10 ng/ml. Following addition of theinterferons, the cells are incubated for 72 hours at 37° C. in ahumidified 5% CO₂ incubator. The number of viable cells in each well isthen determined by the addition of 100 μl of CellTiter-Glo™ (Promega,Cat #G7572). The cells plus CellTiter-Glo™ reagent are mixed for 2minutes and incubated at room temperature in the dark for 30-60 minutes.The amount of luminescent signal for each well is determined using anAnalyst HT instrument.

Analysis:

The EC₅₀ of the IFN-alpha reference and samples were calculated usingthe equation:$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{1 + 10^{{({{{Log}\quad{EC}\quad 50} - X})} \cdot {HillSlope}}}}$

where Bottom is the Y value at the bottom plateau; Top is the Y value atthe top plateau, and Log EC50 is the X value when the response ishalfway between Bottom and Top. The Levenberg-Marquardt method was usedas optimization algorithm.

Example 1 Determination of Surface-Accessible Residues ofInterferon-Alphas

Surface Exposure of Human Interferon-α2a Residues:

Based on the 24 NMR structures of human interferon-alpha 2a reported byKlaus et al., J. Mol. Biol., 274: 661-675 (1997), the fractional ASA ofside chains was calculated. The sequence numbering used below is basedon the mature sequence of the human interferon-alpha 2a protein(identified herein as SEQ ID NO:47). It is noted that this structurecontains two disulphide bridges involving Cys1-Cys98 and Cys29-Cys138,respectively. By computing the ASA and the fractional ASA and taking theaverage of the 24 structures, focusing on the ASA of the side chains, itwas determined that the following residues have more than 25% fractionalASA: D2, L3, P4, Q5, T6, H7, S8, L9, G10, R12, R13, M16, A19, Q20, R22,K23, I24, S25, L26, F27, S28, L30, K31, R33, H34, D35, G37, Q40, E41,E42, G44, N45, Q46, Q48, K49, A50, E51, E58, Q61, Q62, N65, S68, T69,K70, D71, S73, A74, D77, E78, T79, L80, D82, K83, T86, Y89, Q90, N93,D94, E96, A97, V99, I100, Q101, G102, V103, G104, T106, E107, T108,P109, L110, M111, K112, E113, D114, L117, R120, K121, Q124, R125, T127,L128, K131, E132, K133, K134, Y135, S136, P137, C138, A145, M148, R149,S152, L153, N156, Q158, E159, S160, L161, R162, S163, K164 and E165,with position numbering relative to that of the interferon-alpha 2asequence identified herein as SEQ ID NO:47.

The following residues were determined to have on average more than 50%fractional ASA of their side chain: D2, L3, P4, Q5, T6, H7, S8, L9, R12,R13, M16, A19, S25, F27, S28, K31, R33, H34, D35, G37, E41, G44, N45,Q46, Q48, K49, N65, K70, A74, D77, E78, T79, D82, K83, T86, Y89, Q90,N93, D94, I100, Q101, G102, G104, T106, E107, T108, P109, L110, E113,D114, L117, R120, K121, Q124, R125, L128, K131, E132, K134, P137, R149,E159, L161, R162, S163, K164 and E165, with position numbering relativeto that of the interferon-alpha 2a sequence identified herein as SEQ IDNO:47.

Surface Exposure of Residues Corresponding to SEQ ID NO:1:

Owing to an insertion of an amino acid after position 44 of the humaninterferon-alpha 2 subtypes - such as, for example, interferon-alpha 2b(SEQ ID NO:46) and interferon-alpha 2a (SEQ ID NO:47)—in many interferonalpha sequences, including all of the known human interferon alphasequences (apart from the IFN-alpha 2 subtypes) and certain polypeptidesof the invention, the position numbering of the surface-exposed residueswill be shifted by one residue past position number 44 in, for example,the sequences shown in the alignment FIG. 2, relative to the numberingof the sequence denoted hIFNalpha 2b (SEQ ID NO:46).

Based on the above analysis, the following positions, numbered relativeto SEQ ID NO:1, are considered to contain amino acid residues havingmore than 25% fractional ASA: positions 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,13, 16, 19, 20, 22, 23, 24, 25, 26, 27, 28, 30, 31, 33, 34, 35, 37, 40,41, 42, 44, 46, 47, 49, 50, 51, 52, 59, 62, 63, 66, 69, 70, 71, 72, 74,75, 78, 79, 80, 81, 83, 84, 87, 90, 91, 94, 95, 97, 98, 100, 101, 102,103, 104, 105, 107, 108, 109, 110, 111, 112, 113, 114, 115, 118, 121,122, 125, 126, 128, 129, 132, 133, 134, 135, 136, 137, 138, 139, 146,149, 150, 153, 154, 157, 159, 160, 161, 162, 163, 164, 165, and 166.

Likewise, the following positions, again numbered relative to SEQ IDNO:1, are considered contain amino acid residues having on average morethan 50% fractional ASA of their side chain: 2, 3, 4, 5, 6, 7, 8, 9, 12,13, 16, 19, 25, 27, 28, 31, 33, 34, 35, 37, 41, 44, 46, 47, 49, 50, 66,71, 75, 78, 79, 80, 83, 84, 87, 90, 91, 94, 95, 101, 102, 103, 105, 107,108, 109, 110, 111, 114, 115, 118, 121, 122, 125, 126, 129, 132, 133,135, 138, 150, 160, 162, 163, 164, 165, and 166.

Example 2 Antiviral Activites of Interferon-Alpha Polypeptides

Patients with chronic HCV infection have initial viral loads in therange of 10⁴-10⁷ copies of HCV RNA/ml. Upon treatment with IFN-alpha,the viral load characteristically undergoes two distinct log-linearphases of decline reflecting two distinct mechanisms (FIG. 1B). Theinitial drop in viral load occurs in about the first two days and isbelieved to be due to the reduction in rate of virus production byinfected liver cells in the face of the IFN-alpha therapy.

The major technical challenge with HCV is that the virus cannot be grownin vitro and has only recently been cultured in tractable animal models.There are, however, viruses that replicate in vitro which are consideredto be useful surrogates for HCV viral replication. In vitro surrogateassays believed to be predictive of in vivo HCV antiviral activityinclude the assays described in the Materials and Methods section above,which measure the ability of test molecules to protect cells from thecytopathic effect (CPE) of viral infection, using Encephalomyocarditisvirus (EMCV) in the human cell lines HuH7 (hepatoma derived), HeLa(cervical carcinoma derived) and WISH (amniotic tissue derived).

Table 7 below provides relative antiviral activities of variousIFN-alpha polypeptides in representative CPE assays performed asdescribed in the Materials and Methods section above. Significantdifferences in activity were determined by TwoWay ANOVA (factors:sample, experiment) (α=0.05) followed by Bonferroni's post-hoc test formean comparisons (α=0.05) or Fisher's post-hoc test. The symbol (+/−)indicates antiviral activity which is statistically indistinguishablefrom that of the reference interferon; ND, not determined. TABLE 7HuH7/EMCV HeLa/EMCV Antiviral activity Antiviral activity relative torelative to Sample Name SEQ ID NO hulFN-alpha 2b hulFN-alpha 2b B9X14EC4SEQ ID NO: 1 >8x >8x 14EPI15 SEQ ID NO: 10 >8x >8x 14EPI16 SEQ ID NO:11 >8x >8x 14EPI17 SEQ ID NO: 12 >8x >8x 14EPI18 SEQ ID NO: 13 >8x >8x14EPI19 SEQ ID NO: 14 >8x >8x 14EPI20 SEQ ID NO: 15 >8x >8x 14EPI21 SEQID NO: 16 >8x >8x 14EPI25 SEQ ID NO: 20 >8x >8x 14EPI32 SEQ ID NO:27 >8x >8x 14EPI33 SEQ ID NO: 28 >8x >8x 14EPI34 SEQ ID NO: 29 >8x >8x25EPI19 SEQ ID NO: 38 >8x >8x 14EPI22 SEQ ID NO: 17 4x to 8x >8x 14EPI26SEQ ID NO: 21 4x to 8x >8x 14EPI29 SEQ ID NO: 24 4x to 8x >8x 14EPI31SEQ ID NO: 26 4x to 8x >8x 14EPI23 SEQ ID NO: 18 4x to 8x 4x to 8x14EPI24 SEQ ID NO: 19 4x to 8x 4x to 8x 25EPI08 SEQ ID NO: 36 4x to 8x4x to 8x 25EPI26 SEQ ID NO: 41 4x to 8x 4x to 8x 14EPI42 SEQ ID NO: 354x to 8x 2x to 4x 25EPI18 SEQ ID NO: 37 4x to 8x 2x to 4x 25EPI25 SEQ IDNO: 40 4x to 8x 2x to 4x 14EPI30 SEQ ID NO: 25 2x to 4x 4x to 8x 25EPI28SEQ ID NO: 43 2x to 4x 2x to 4x 14EPI27 SEQ ID NO: 22 2x to 4x 2x to 4x14EPI35 SEQ ID NO: 30 2x to 4x ND 14EPI36 SEQ ID NO: 31 2x to 4x ND14EPI37 SEQ ID NO: 32 2x to 4x ND 14EPI38 SEQ ID NO: 33 2x to 4x ND25EPI20 SEQ ID NO: 39 2x to 4x 0.5x to 2x   14EPI28 SEQ ID NO: 23 0.5xto 2x   0.5x to 2x   25EPI27 SEQ ID NO: 42 0.1x to 0.5x 0.1x to 0.5xIFN-alpha SEQ ID NO: 58 2x to 4x 4x to 8x Con1 IFN-alpha 2a SEQ ID NO:47 0.5x to 2x   0.5x to 2x   IFN-alpha 2b SEQ ID NO: 46 1 (ref) 1 (ref)

Example 3 T_(H)1 Activites of Interferon-Alpha Polypeptides

The T_(H)1 assay was designed to monitor the enhancement ofdifferentiation of naïve T cells into T_(H)1 cells by IFN-alpha asdetermined by the amount of IFN-gamma detected in the culturesupernatants. Table 8 provides relative T_(H)1 activities of variousIFN-alpha polypeptides assayed as described in the Materials and Methodssection above. TABLE 8 Fold over Sample SEQ ID NO IFN-alpha 2a 14Epi18SEQ ID NO: 13 >30x 25Epi19 SEQ ID NO: 38 >30x IFN-alpha Con1 SEQ ID NO:58 10x to 15x IFN-alpha 2a SEQ ID NO: 47 1 (ref)

Example 4 Antiproliferative Activities of Interferon-Alpha Polypepyides

IFN-alpha inhibits proliferation of many cell types, although theantiproliferative effects often occur at higher doses than are requiredfor the antiviral response. Daudi cells are a human derivedEVB-transformed B cell line that is IFN-alpha sensitive. This IFN-alpharesponsive cell line serves as a useful probe of the antiproliferativeeffects of the IFN-alpha polypeptides of the invention. Furthermore,antiproliferative activity of IFN-alpha on megakaryocytes andneutrophils at high dose is believed to contribute to thrombocytopeniaand neutropenia, respectively. The Daudi antiproliferation assay mayserve as a useful surrogate assay for antiproliferative effects on theseother lymphoid cell types.

Table 9 provides relative antiproliferative activities of variousIFN-alpha polypeptides assayed as described in the Materials and Methodssection above, expressed as fold antiproliferative activity relative tohuIFN-alpha 2a (EC₅₀ huIFN-alpha 2a/EC₅₀ sample). Under the assayconditions employed, antiproliferative activities at least 0.01× of thereference interferon were detectable. TABLE 9 Fold activity sample nameSEQ ID NO: vs. IFNα-2a B9X14EC4 SEQ ID NO: 1 0.8x to 0.5x  14Epi15 SEQID NO: 10 0.25x to 0.1x  14Epi17 SEQ ID NO: 12 0.25x to 0.1x  14Epi18SEQ ID NO: 13 0.8x to 0.5x  14Epi19 SEQ ID NO: 14 0.5x to 0.25x 14Epi20SEQ ID NO: 15 0.5x to 0.25x 14Epi21 SEQ ID NO: 16 0.25x to 0.1x  14Epi22SEQ ID NO: 17 0.25x to 0.1x  14Epi23 SEQ ID NO: 18 0.25x to 0.1x 14Epi24 SEQ ID NO: 19 0.5x to 0.25x 14Epi25 SEQ ID NO: 20 0.5x to 0.25x14Epi26 SEQ ID NO: 21 0.5x to 0.25x 14Epi27 SEQ ID NO: 22 0.5x to 0.25x14Epi28 SEQ ID NO: 23 0.8x to 0.5x  14Epi29 SEQ ID NO: 24 0.25x to 0.1x 14Epi30 SEQ ID NO: 25 0.5x to 0.25x 14Epi31 SEQ ID NO: 26 0.5x to 0.25x14Epi32 SEQ ID NO: 27 1.2x to 0.8x  14Epi33 SEQ ID NO: 28 0.8x to 0.5x 14Epi34 SEQ ID NO: 29 0.8x to 0.5x  14Epi35 SEQ ID NO: 30 0.8x to 0.5x 14Epi36 SEQ ID NO: 31 0.5x to 0.25x 14Epi37 SEQ ID NO: 32 0.8x to 0.5x 14Epi38 SEQ ID NO: 33 0.8x to 0.5x  14Epi42 SEQ ID NO: 35 0.5x to 0.25x25Epi08 SEQ ID NO: 36 0.5x to 0.25x 25Epi18 SEQ ID NO: 37 0.25x to 0.1x 25Epi19 SEQ ID NO: 38 0.8x to 0.5x  25Epi20 SEQ ID NO: 39 0.25x to 0.1x 25Epi25 SEQ ID NO: 40 0.25x to 0.1x  25Epi26 SEQ ID NO: 41 0.25x to0.1x  25Epi27 SEQ ID NO: 42 0.5x to 0.25x 25Epi28 SEQ ID NO: 43 0.5x to0.25x 25Epi29 SEQ ID NO: 44 0.8x to 0.5x  IFN-alpha Con1 SEQ ID NO: 581.2x to 0.8x  IFN-alpha 2a SEQ ID NO: 47 1x (ref)

Example 5 Pegylation of Interferon-Alpha Polypeptides

The following provides exemplary procedures for preparing conjugates ofthe invention.

Cys-PEGylation

A polypeptide of the invention which contains a free cysteine may becysteine-PEGylated as follows. The polypeptide is first partiallyreduced with an equimolar concentration of TCEP(Triscarboxyethylphosphine) at 4° C. for 30 min in 50 mM MES, 100 mMNaCl, pH 6.0. The reduced polypeptide is then reacted with a 4 foldmolar excess of mPEG-MAL reagent (with a PEG moiety such as a 20 kDa or30 kDa linear mPEG, or a 40 kDa branched mPEG2) for lh at 4° C. underthe same conditions. The PEGylated reaction mixture is loaded on to aSP-Sepharose HP column equilibrated with 50 mM MES, pH 6.0, 100 mM NaCl.After a 10 CV (column volume) wash step a gradient from 0-600 mM NaCl isapplied to fractionate the PEGylated and unPEGylated fractions.Fractions are collected and aliquots are analyzed by SDS-PAGE. Fractionscontaining monoPEGylated species are pooled and formulated for assaysfor interferon-alpha activity as described above.

N-Terminal PEGylation

N-terminal PEGylation with mPEG-butyraldehyde (having a PEG moiety suchas a 20 kDa or 30 kDa linear mPEG, or a 40 kDa branched mPEG2) may beperformed at 4° C. using a 5 fold molar excess of PEGreagent:polypeptide for 4-8 h in 50 mM MES, pH 5.5, 100 mM NaCl, and 20mM sodium cyanoborohydride, or in 50 mM sodium acetate, pH 5.5, 100 mMNaCl and 20 mM sodium cyanoborohydride. Conjugates are isolated bychromatography over a SP-Sepharose HP column equilibrated in 50 mM MESpH 5.5, 100 mM NaCl, using a gradient from 100-600 mM NaCl in 50 mM MESpH 5.5. Fractions containing monoPEGylated species are pooled andformulated for assays for interferon-alpha activity as described above,and may be further characterized by, for example, using analytical HPLC,amino acid analysis and/or MALDI-TOF mass spectrometry.

Lysine PEGylation

In general, lysine-PEGylation was performed using a procedureessentially as described by Foser et al. (2003) Protein Expression &Purification 30:78-87. Briefly, the polypeptide was reacted with 40 kDamPEG2-NHS at about a 3:1 to 5:1 molar ratio of mPEG2-NHS:polypeptide in50 mM borate buffer, pH 9.0, at 4° C. for about 1 hr to overnight.Conjugates were isolated by cation exchange chromatography. Fractionscontaining monoPEGylated species were pooled and formulated forinterferon-alpha activity assays as described above. MonoPEGylatedIFN-alpha conjugates prepared according to the above procedure exhibitedantiviral activity in the HeLa-EMCV assay, albeit lower than that of theparent unPEGylated polypeptides.

In some instances, the monoPEGylated pool obtained as described abovewas further analyzed by cation exchange HPLC using a Polysulfoethyl Acolumn (2.1 mmID×200 mmL, 5 m, 1000 A°, from PolyLC) to characterize thePEG-isomer profile. The mobile phases were buffer A: 5 mM KH₂PO4, 40%1-propanol, 1% diethylene glycol, pH 3.0 and bufferB: same as A with 200mM NaCl. The PEG positional isomers were separated using a gradient from50%-75% mobile phase B over 80 min at 0.1 ml/min, 20° C. with detectionat 214 nm.

FIG. 8 shows the separation by cation exchange HPLC of the monoPEGylatedpool obtained from the reaction of interferon-alpha polypeptide B9x14EC4with 40 kDa mPEG2-NHS according to the above procedure. The peaks werecollected and analyzed on SDS-PAGE (FIG. 9). Lanes 2 and 9 of FIG. 9shows that the sample prior to application onto the HPLC column containspredominantly monoPEGylated protein. Peaks 1-4 (lanes 3-6 of FIG. 9)correspond to the small proportion of diPEGylated IFN in themonoPEGylated pool. Minor peak 5 (lane 7 of FIG. 9) containsapproximately equal proportions of diPEGylated, monoPEGylated and alower molecular weight species. Peaks 6-11 (lanes 10-15 of FIG. 9)correspond to positional isomers of the monoPEGylated IFN polypeptide.

Polypeptide 14epi18 (SEQ ID NO:13) was lysine-PEGylated as follows. Theeluate of the Q-Sepharose chromatography step (obtained according to theMaterials and Methods section above) was concentrated to c<2 mg/ml. ForPEGylation the buffer was exchanged to 50 mM sodium borate, pH 9.0. Thepolypeptide was reacted with 40 kDa mPEG2-NHS essentially as describedby Foser et al. (2003; Protein Expression & Purification 30:78-87) at amolar ratio of PEG reagent over protein of 4:1 or 5:1. The PEGylationreaction was allowed to proceed at 4° C. for 1.5 h. MonoPEGylated14epi18 was purified from the reaction mixture by cation exchangechromatography. Elution was performed in three steps using increasingsalt concentrations up to 1 M NaCl to separate oligoPEGylated,monoPEGylated and non-PEGylated species. The monoPEGylated 14epi18 peakwas formulated at a concentration of 0.33 mg/ml for further analysis.The monoPEGylated 14epi18 composition so obtained yielded a populationof positional isomers predominantly monoPEGylated at Lys122 and Lys135.

Polypeptide 25epi19 (SEQ ID NO:38) was lysine-PEGylated as follows. Theeluate of the Q-Sepharose chromatography step (obtained according to theMaterials and Methods section above) was concentrated to c<2 mg/ml. ForPEGylation the buffer was exchanged to 50 mM sodium borate, pH 9.0. Thepolypeptide was reacted with 40 kDa mPEG2-NHS essentially as describedby Foser et al. (2003; Protein Expression & Purification 30:78-87) at amolar ratio of PEG reagent over protein of 4:1. The PEGylation reactionwas allowed to proceed at 4° C. overnight. MonoPEGylated 25epi19 waspurified from the reaction mixture by cation exchange chromatography.Elution was performed in three steps using increasing saltconcentrations up to 750 mM NaCl to separate oligoPEGylated,monoPEGylated and non-PEGylated species. The monoPEGylated 25epi19 peakwas concentrated in the elution buffer to <2 mg/ml and formulated forfurther analysis. The monoPEGylated 25epi19 composition so obtainedyielded a population of positional isomers predominantly monoPEGylatedat Lys31, Lys122 and Lys135.

MonoPEGylated 14epi18 and monoPEGylated 25epi19 prepared as describedabove both exhibited greater than 10-fold higher antiviral activity thanmonoPEGylated IFN-alpha 2a in the HeLa-EMCV antiviral assay.

Example 6 Differential Scanning Calorimetry

Differential scanning calorimetry (DSC) experiments were performed toestimate the stability of some polypeptides of the invention. Samples ofprotein at concentrations from 100 to 325 μg/ml were buffer-exchangedinto phosphate buffered saline (PBS) and subjected to DSC using aMicroCal VP-DSC (MicroCal, Northampton, Mass.). The melting temperature(T_(m)) was estimated from the plot of heat capacity (C_(p)) vstemperature. Estimated T_(m)s are: B9X14EC4 (SEQ ID NO:1) 58.4° C.;14epi16 (SEQ ID NO:11) 64.2° C.; and 14epi18 (SEQ ID NO:13) 59.5° C. Thesubstitution of a buried phenylalanine residue at position 48 (in SEQ IDNO:1) for either a leucine (in SEQ ID NO:11) or an alanine (in SEQ IDNO:13) appears to have no detrimental effect on the stability of thepolypeptide.

Example 7 In Vivo Assays

Measurement of Pharmacokinetic (PK) and Pharmacodynamic (PD) Profiles ofa Polypeptide or Conjugate of the Invention

Measurement of biological or serum half-life may be carried out in anumber of ways described in the literature. For example, biologicalhalf-life may be determined using an ELISA method to detect serum levelsof interferon-alpha after e.g. subcutaneous or intramuscularadministration. Use of an ELISA method to determine the pharmacokineticsof interferon-alpha administered subcutaneously is e.g. described byRostaing et al. (1998), J. Am. Soc. Nephrol. 9(12): 2344-48. Merimsky etal. (1991), Cancer Chemother. Pharmacol. 27(5); 406-8, describe thedetermination of the serum level of an interferon-alpha administeredintramuscularly.

For example, the pharmacokinetic (PK) and pharmacodynamic (PD) profilesof a conjugate of the invention may be studied in cynomolgus monkeys(Macaca fascicularis) according to the following procedure. Three tofour year old female animals with an average weight of 5-6 kg are usedin the study. Animals are acclimatized for a minimum of 6 weeks betweenarrival and start of treatment with a minimum period of 3 weeksacclimatization to the study housing conditions. They are housed singlyin stainless-steel mesh cages (at least 540×810×760 mm) and are kept onan expanded complete commercial primate diet (100 grams/animal/day)analyzed for the absence of chemical and bacterial contaminants. Inaddition, animals receive fruit daily (apple or banana). Animals arefasted before all procedures requiring anesthesia.

Three days before study initiation and on the day of study initiationprior to administration of the compounds, blood samples are collectedfrom all animals. Throughout the study, blood samples (1 ml of wholeblood) are withdrawn from a femoral or cephalic/saphenous vessel of theunanesthetised manually restrained animal. The normal feeding regime ismaintained during collection. Samples are collected in tubes withoutanticoagulant, followed by centrifugation at 3000 rpm (about 1760 g) for10 minutes at 4° C. The serum samples are then separated into twoaliquots (approximately 200 μl each) and stored at −20° C.

Prior to initiation of the study, the conjugate of the invention isformulated in 20 mM sodium acetate (pH 6.0) and 140 mM sodium chloride.At study initiation, the conjugate is administered once per animal ateither 30 mcg conjugate/kg animal weight (four animals), or 300 mcg/kg(four animals), using a sterile syringe and needle introducedsubcutaneously after local disinfection with an aqueous solution ofethyl alcohol. Blood samples are collected at the following time pointsafter administration of the conjugate: 4 hours, 8 hours, 12 hours, 24hours, 48 hours, 72 hours, 144 hours, 240 hours, 336 hours, 456 hours,552 hours, 600 hours, 624 hours, 648 hours, 672 hours, 696 hours, 720hours and 744 hours.

The animals are observed daily to detect any clinical signs or reactionto treatment. Injection sites are visually assessed daily in order toevaluate local tolerance. A detailed clinical examination is performedbefore the initiation of treatment and at study termination.

The concentration of conjugate in the isolated serum samples aredetermined after completion of the study, using an ELISA assay. The invivo half-life of the conjugate in cynomolgus monkeys is then determinedbased on the serum concentration of the compounds at the specified timepoints.

The pharmacodynamic response in cynomolgus monkeys to treatment with aconjugate of the invention is evaluated by measuring the levels of2′,5′-oligoadenylate synthetase and neopterin in each of the serumsamples, using a commercially available RIA (Eiken Chemical Co., Tokyo)and a commercially available ELISA (IBL Immuno Biological Laboratories,Hamburg.), respectively. Based on the measured concentrations of thesetwo biomarkers for interferon activity in the serum samples, the areaunder the curve (AUC) is determined to quantify the in vivo activity ofeach compound at each of the two dosages.

Determining In Vitro Immunogenicity

Reduced immunogenicity of a polypeptide or conjugate of the inventionrelative to a reference molecule can be determined by use of an ELISAmethod measuring the immunoreactivity of the molecule relative to areference molecule or preparation, typically a known interferon-alphaprotein. The ELISA method is based on antibodies from patients treatedwith the reference protein. The immunogenicity is considered to bereduced when the polypeptide or conjugate of the invention has astatistically significant lower response in the assay than the referencemolecule or preparation.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. For example, all thetechniques and apparatus described above may be used in variouscombinations. All publications, patents, patent applications, and/orother documents cited in this application are incorporated herein byreference in their entirety for all purposes to the same extent as ifeach individual publication, patent, patent application, and/or otherdocument were individually indicated to be incorporated herein byreference in its entirety for all purposes.

1.-7. (canceled)
 8. An isolated or recombinant polypeptide comprising asequence which (a) differs in 0 to 16 amino acid positions from SEQ IDNO:13 and (b) comprises Ala or Leu at position 48; which polypeptideexhibits antiviral activity.
 9. The polypeptide of claim 8, whichsequence comprises Ala position
 48. 10. The polypeptide of claim 8,comprising a sequence which differs from SEQ ID NO:13 in 0 to 8 aminoacid positions.
 11. An isolated or recombinant polypeptide comprisingthe sequence SEQ ID NO:13, optionally further comprising a methionine atthe N-terminus, which polypeptide exhibits antiviral activity. 12.-19.(canceled)
 20. An isolated or recombinant polypeptide comprising asequence which (a) differs in 0 to 16 amino acid positions from SEQ IDNO:38 and (b) comprises Ala or Leu at position 48; which polypeptideexhibits antiviral activity.
 21. The polypeptide of claim 20, whichsequence comprises Ala at position
 48. 22. The polypeptide of claim 20,comprising a sequence which differs from SEQ ID NO:38 in 0 to 8 aminoacid positions.
 23. An isolated or recombinant polypeptide comprisingthe sequence SEQ ID NO:38 or the sequence of a variant thereof whereinthe variant differs in 1 to 2 amino acid positions from SEQ ID NO:38,optionally further comprising a methionine at the N-terminus, whichpolypeptide exhibits antiviral activity.
 24. The polypeptide of claim 8,11, 20, 23, or 77, wherein the antiviral activity of the polypeptide isequal to or greater than the antiviral activity of huIFN-alpha 2b orhuIFN-alpha 2a.
 25. The polypeptide of claim 24, wherein the antiviralactivity of the polypeptide is at least two-fold greater than theantiviral activity of huIFN-alpha 2b or huIFN-alpha 2a.
 26. Thepolypeptide of claim 8, 11, 20, 23, or 77, wherein the polypeptideexhibits a ratio of antiviral activity/antiproliferative activity atleast two-fold greater than the ratio of antiviralactivity/antiproliferative activity exhibited by huIFN-alpha 2b orhuIFN-alpha 2a.
 27. A conjugate comprising (a) the polypeptide of claim8, 11, 20, 23, or 77, and (b) a non-polypeptide moiety covalentlyattached to the polypeptide, which conjugate exhibits antiviralactivity.
 28. The conjugate of claim 27, comprising at least twonon-polypeptide moieties covalently attached to the polypeptide.
 29. Theconjugate of claim 27, comprising a non-polypeptide moiety covalentlyattached to a cysteine residue.
 30. The conjugate of claim 27,comprising a non-polypeptide moiety covalently attached to a lysineresidue or to the N-terminal amino group.
 31. The conjugate of claim 27,comprising a non-polypeptide moiety covalently attached to a lysineresidue.
 32. The conjugate of claim 27, comprising a non-polypeptidemoiety attached to the N-terminal amino group.
 33. The conjugate ofclaim 27, wherein the non-polypeptide moiety is a polymer.
 34. Theconjugate of claim 33, wherein the polymer is a polyethylene glycol. 35.The conjugate of claim 34, wherein the polyethylene glycol is a 40 kDampEG2 moiety covalently attached to a lysine residue.
 36. The conjugateof claim 27, wherein the non-polypeptide moiety is a sugar.
 37. Theconjugate of claim 36, wherein the sugar is attached to anN-glycosylation site of the polypeptide.
 38. A conjugate comprising (a)a polypeptide comprising the sequence SEQ ID NO:13, optionally furthercomprising a methionine at the N-terminus; and (b) a PEG moietycovalently attached to a lysine residue of the polypeptide, whichconjugate exhibits antiviral activity.
 39. The conjugate of claim 38,wherein the PEG moiety is a 40 kDa mPEG2 moiety.
 40. The conjugate ofclaim 39, wherein the 40 kDa mPEG2 moiety is covalently attached to alysine residue selected from Lys122 and Lys135.
 41. A conjugatecomprising (a) a polypeptide comprising the sequence SEQ ID NO:38 or thesequence of a variant thereof wherein the variant differs in 1 to 2amino acid positions from SEQ ID NO:38, optionally further comprising amethionine at the N-terminus; and (b) a PEG moiety covalently attachedto a lysine residue of the polypeptide, which conjugate exhibitsantiviral activity.
 42. The conjugate of claim 41 or 79, wherein the PEGmoiety is a 40 kDa mPEG2 moiety.
 43. The conjugate of claim 42, whereinthe 40 kDa mPEG2 moiety is covalently attached to a lysine residueselected from Lys31, Lys122, and Lys135.
 44. A composition comprising(a) a conjugate comprising a polypeptide comprising the sequence SEQ IDNO:13, optionally further comprising a methionine at the N-terminus, anda 40 kDa mPEG2 moiety covalently attached to Lys122; and (b) a conjugatecomprising a polypeptide comprising the sequence SEQ ID NO:13,optionally further comprising a methionine at the N-terminus, and a 40kDa mPEG2 moiety covalently attached to Lys135; which compositionexhibits antiviral activity.
 45. A composition comprising (a) aconjugate comprising a polypeptide comprising the sequence SEQ ID NO:38,optionally further comprising a methionine at the N-terminus, and a 40kDa mPEG2 moiety covalently attached to Lys31; (b) a conjugatecomprising a polypeptide comprising the sequence SEQ ID NO:38,optionally further comprising a methionine at the N-terminus, and a 40kDa mPEG2 moiety covalently attached to Lys122; and (b) a conjugatecomprising a polypeptide comprising the sequence SEQ ID NO:38,optionally further comprising a methionine at the N-terminus, and a 40kDa mPEG2 moiety covalently attached to Lys135; which compositionexhibits antiviral activity.
 46. A composition comprising thepolypeptide of claim 8, 11, 20, 23, or 77, and a pharmaceuticallyacceptable excipient.
 47. A composition comprising the conjugate ofclaim 38, 41, or 79, and a pharmaceutically acceptable excipient. 48.The composition of claim 44 or 45 further comprising a pharmaceuticallyacceptable excipient.
 49. An isolated or recombinant polynucleotidecomprising a nucleic acid sequence which encodes the polypeptide ofclaim 8, 11, 20, 23, or
 77. 50. The polynucleotide of claim 49,comprising a nucleic acid sequence which encodes a polypeptidecomprising the sequence SEQ ID NO:13.
 51. The polynucleotide of claim50, comprising a nucleic acid sequence selected from SEQ ID NO:62, SEQID NO:63, SEQ ID NO:64, and SEQ ID NO:89.
 52. The polynucleotide ofclaim 49, comprising a nucleic acid sequence which encodes a polypeptidecomprising the sequence SEQ ID NO:38.
 53. The polynucleotide of claim52, comprising a nucleic acid sequence selected from SEQ ID NO:80, SEQID NO:81, SEQ ID NO:82, and SEQ ID NO:90.
 54. A host cell comprising thepolynucleotide of claim
 49. 55. A vector comprising the polynucleotideof claim
 49. 56. The vector of claim 55, which is an expression vectorcomprising the polynucleotide operably linked to a promoter.
 57. A hostcell comprising the vector of claim
 56. 58. A composition comprising thepolynucleotide of claim 49 and an excipient.
 59. A method for preparingthe polypeptide of claim 8, 11, 20, 23, or 77, the method comprising:providing a culture comprising a host cell, the host cell comprising anexpression vector comprising a promoter operably linked to apolynucleotide, the polynucleotide comprising a nucleic acid sequencewhich encodes the polypeptide, culturing the culture under conditionswhich permit expression of the polypeptide, and recovering thepolypeptide.
 60. The method of claim 59, wherein the host cell is aglycosylating host cell.
 61. The method of claim 59, wherein the hostcell is a bacterial host cell.
 62. The method of claim 59, wherein thebacterial host cell is E. coli.
 63. The method of claim 62, wherein thestep of recovering the polypeptide comprises: (a) isolating inclusionbodies (IB) comprising the polypeptide; (b) solubilizing the IB, therebyobtaining unfolded polypeptide; (c) refolding the unfolded polypeptide,thereby obtaining refolded polypeptide; and (d) purifying the refoldedpolypeptide.
 64. A method for preparing a conjugate, the methodcomprising (i) providing the polypeptide of claim 8, 11, 20, 23, or 77,and (ii) attaching at least one non-polypeptide moiety to thepolypeptide, wherein the resulting conjugate exhibits antiviralactivity.
 65. The method of claim 64, wherein the step of providing thepolypeptide comprises: providing a culture comprising a host cell, thehost cell comprising an expression vector comprising a promoter operablylinked to a polynucleotide, the polynucleotide comprising a nucleic acidsequence which encodes the polypeptide, culturing the culture underconditions which permit expression of the polypeptide, and recoveringthe polypeptide.
 66. The method of claim 65, wherein the host cell is aglycosylating host cell or a bacterial host cell.
 67. The method ofclaim 66, wherein the bacterial host cell is E. coli.
 68. The method ofclaim 67, wherein the step of recovering the polypeptide comprises: (a)isolating inclusion bodies (IB) comprising the polypeptide; (b)solubilizing the IB, thereby obtaining unfolded polypeptide; (c)refolding the unfolded polypeptide, thereby obtaining refoldedpolypeptide; and (d) purifying the refolded polypeptide.
 69. The methodof claim 64, wherein the step of attaching the at least onenon-polypeptide moiety to the polypeptide comprises: reacting thepolypeptide with 40 kDa mPEG2-NHS at a molar ratio ofmPEG2-NHS:polypeptide of about 3:1 to 5:1 at pH 9 and 4° C. for 1 hourto overnight.
 70. A method for reducing the number of copies of a virusin cells infected with the virus, the method comprising: administeringthe conjugate of claim 27 to the cells in an amount effective to reducethe number of copies of the virus in the cells, thereby reducing thenumber of copies of the virus in said cells.
 71. A method for reducingthe level of HCV RNA in the serum of a patient infected with HCV,comprising administering to the patient the conjugate of claim 27 in anamount effective to reduce the level of HCV RNA compared to the HCV RNAlevel present prior to the start of treatment.
 72. A method for reducingthe level of HBV DNA in serum of a patient infected with HBV, comprisingadministering to the patient the conjugate of claim 27 in an amounteffective to reduce the level of HBV DNA compared to the HBV DNA levelpresent prior to the start of treatment.
 73. A method for reducing thelevel of HIV RNA in serum of a patient infected with HIV, comprisingadministering to the patient the conjugate of claim 27 in an amounteffective to reduce the level of HIV RNA compared to the HIV RNA levelpresent prior to the start of treatment.
 74. The polypeptide of claim 8,comprising a sequence which differs from SEQ ID NO:13 in 0 to 4 aminoacid positions.
 75. The polypeptide of claim 22, comprising a sequencewhich differs from SEQ ID NO:38 in 0 to 4 amino acid positions.
 76. Thepolypeptide of claim 23, comprising the sequence SEQ ID NO:38.
 77. Anisolated or recombinant polypeptide comprising the sequence SEQ ID NO:39or the sequence of a variant thereof wherein the variant differs in 1 to3 amino acid positions from SEQ ID NO:39, optionally further comprisinga methionine at the N-terminus, which polypeptide exhibits antiviralactivity.
 78. The conjugate of claim 41, wherein the polypeptidecomprises the sequence SEQ ID NO:38.
 79. A conjugate comprising (a) apolypeptide comprising the sequence SEQ ID NO:39 or the sequence of avariant thereof wherein the variant differs in 1 to 3 amino acidpositions from SEQ ID NO:39, optionally further comprising a methionineat the N-terminus; and (b) a PEG moiety covalently attached to a lysineresidue of the polypeptide, which conjugate exhibits antiviral activity.